BD PharMingen

Rat Immunology From Genes to Proteins to Cells

BD Biosciences www.bdbiosciences.com • Biometric Imaging • Immunocytometry Systems • Microbiology Systems • Transduction Laboratories • Clontech • Labware • PharMingen

GENES CELLS

PROTEINS

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Cells B Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 NEW Rat FcBlock™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 T Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Monocytes/Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Cell Surface Adhesion Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 MHC Haplotype Chart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Proteins Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Detection of Intracellular Cytokines by Flow Cytometry . . . . . . . . . . . . . 17 BD OptEIA™ Rat Cytokine ELISA Sets . . . . . . . . . . . . . . . . . . . . . . . . . 19 OptEIA™ Sets General ELISA Reagents . . . . . . . . . . . . . . . . . . . . . . . . . 20 Cell Biology: Apoptosis and Neurobiology . . . . . . . . . . . . . . . . . . . . . . . 22

Genes BD RiboQuant™ Ribonuclease Protection Assay (RPA) Systems . . . . . . . 26

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Introduction The rat is the second most frequently used animal, after the mouse, in the field of immunological research. Traditionally, rats have been used as experimental animals to study behavior, physiology, and pathology, including tumor biology, transplantation, and toxicology. Together with mice and guinea pigs, rats belong to the largest order of mammals, the Rodentia. The laboratory rat was developed from the brown rat, Rattus norvegicus, at the beginning of the twentieth century, and most laboratory rat strains originate from animals held at the Wistar Institute of Anatomy and Biology in Philadelphia, USA. A large number of genetically welldefined inbred laboratory rat strains are now available for experimental research. In addition, a variety of congenic, mutant and recombinant rat strains of immunological interest have been developed in recent years, further expanding the possibilities of experimentation. These strains include nude (athymic) rats, rats that spontaneously develop diabetes, and pairs of rats congenic for MHC class I (RT1A) or II (RT1B and RT1D) haplotypes, immunoglobulin kappa light chain (Igκ-1) allotypes or the leukocyte common alloantigen (RT7). BD PharMingen offers antibodies that recognize polymorphic and non-polymorphic epitopes of those alloantigen systems. (Please see the Rat MHC Haplotype chart on page 11.) Although rats are small animals, they are big enough to allow a wide variety of experimental manipulations. Their comparably larger size, than mice, offers unique possibilities for the application of microsurgical techniques, therefore rats are often

the species of premier choice for transplantation studies. For example, the recent technology to induce indefinite allograft survival by intrathymic injection of alloantigen was first developed in rats. Rats are also used frequently for the generation of hybridomas and monoclonal antibodies. There are many rat autoimmune disease models which develop spontaneously after immunization with appropriate antigens, after chemical treatments, or upon introduction of a transgene. Some examples of frequently used rat models of human autoimmune diseases are: • Insulin-dependent diabetes mellitus (IDDM). Diabetes-prone bio-breeding (DP-BB) rats spontaneously develop autoimmune insulin-dependent diabetes and thyroiditis at very high frequency. Hyperglycemia develops when rats are between 50 and 90 days old and is associated with infiltration of the islets of Langerhans and selective destruction of pancreatic β cells. • Multiple sclerosis. A widely used animal model for multiple sclerosis is experimental autoimmune encephalomyelitis (EAE). EAE can be induced relatively easily in Lewis rats by inoculation with homogenized central nervous system myelin or purified myelin antigens together with incomplete Freund's adjuvant. Both acute and chronic relapsing EAE models have been developed.

• Glomerulonephritis. Injection of BN rats with multiple low doses of HgCl2 leads to the development of chemically-induced autoimmune glomerulonephritis associated with proteinurea and depositions of autoantibodies to the renal glomerular basement membrane. Many monoclonal antibodies to rat cell surface antigens and soluble factors have been developed in the last twenty years, and their number is rapidly growing. The availability of these antibodies greatly facilitates studies of the immune system in the rat. Many homologues of human Cluster of Differentiation (CD) antigens have been cloned in the rat allowing detailed analysis of relevant molecules of immunological importance. Undoubtedly, experimentation in the rat will continue to be a critical tool in biomedical research. Here, we have summarized selected reagents and techniques available from BD Pharmingen for immunological research in the rat animal model.

• Rheumatoid arthritis. Immunization of rats with mycobacteria suspended in oil results in the development of adjuvant arthritis, an extensively used animal model of rheumatoid arthritis in humans.

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B Lymphocytes

104 CD45RA-PE (OX-33)

103

CD45RA-PE (OX-33)

CD80-PE (3H5)

B

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Figure 1

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B cells generate the humoral immune response and can function as antigen presenting cells for T cell

CD45RA-FITC (OX-33)

activation and thus also play a role in cell mediated immune response. One major difference between rat B cells and those in the human or mouse is that in the rat a CD5+ subset of B cells has not yet been described. Mature B cells in the rat can easily be distinguished from other leukocytes either on the basis of their expression of sIg or on the basis of their expression of CD45 isoforms unique to B cells. These B cell isoforms of CD45 include the CD45R isoform recognized by HIS24 mAb and CD45RA recognized by OX-33 mAb.

103

CD80-PE (3H5)

A

B and T cells comprise the two major populations of small lymphocytes. B cells mature in the bone marrow and are defined by the expression of the B cell receptor complex (BCR). The BCR consists of membrane bound antibody molecules (sIg or mIg) complexed with the Igα (CD79a) and Igβ (CD79b) molecules. While sIg recognizes and binds antigen, CD79a and CD79b participate in BCR signal transduction.

102

PROTEINS

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CELLS

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GENES

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CD45RA-FITC (OX-33)

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RT1A-FITC (OX-18)

Figure 4

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CD45RA-PE (OX-33)

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CD45RA-PE (OX-33)

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Figure 3

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Isotype Control-FITC (G155-178)

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Figure 1,2. Activation-induced changes in the level of expression of the CD80 antigen on fresh splenocytes (A) and cells treated in vitro for 72 hours with lipopolysaccharide (LPS) (B). Cells were stained simultaneously with CD45RAFITC (OX-33) and CD80-PE (3H5).

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Figure 2

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RT1B-FITC (OX-6)

Ig light chain κ-FITC (MRK-1)

Figure 5

Figure 6

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GENES

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CD45RA-PE (OX-33)

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CD45RA-PE (OX-33)

CELLS

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CD5-FITC (OX-19)

IgM-FITC (G53-239)

Figure 7

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CD45RA-PE (OX-33)

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Figure 8

100

101 103 102 CD32-FITC (D34-485)

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Figure 9

CD32 (D34-485) on paraffinembedded spleen

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CD45RA-PE (OX-33)

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Figure 10

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Marginal Zone B Cell Marker-FITC (HIS57)

Marginal Zone B Cell marker (HIS57) on paraffin-embedded spleen

Figure 11

Figure 12

Figures 3-12. Flow cytometry profiles of rat splencytes using CD45RA-PE (OX-33) antibody to separate B cells from other blood leukocytes. Cells were also stained with a panel of FITC-conjugated antibodies that react with cell surface proteins of interest on B cells (as noted on the x-axis). When available, the same antibody (in purified format) was used for immunohistochemical staining of rat tissue sections.

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PROTEINS

While CD64 binds monomeric IgG, CD32 and CD16 bind to aggregated IgG. In addition to Mouse FcBlock™, anti-mouse CD32/16, BD PharMingen now offers Rat FcBlock™ (anti-rat CD32) capable of blocking FcγRmediated binding of aggregated IgG to FcγR bearing cells.

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CD45RA-PE (OX-33)

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CD45RA-PE (OX-33)

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Cells stained after pre-incubation with purified isotype control (no Rat FcBlock™ present)

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Ms IgG1, κ-FITC (A112-2)

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NKR-P1A-FITC (10/78)

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Figure 14

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CD45RA-PE (OX-33)

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Cells stained after pre-incubation with Rat FcBlock™ (anti-rat CD32, D34-485)

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PROTEINS

CD45RA-PE (OX-33)

CELLS

NEW Rat FcBlock™ B cells also express Fcγ receptors (FcγR) belonging to the immunoglobulin superfamily. The major Fcγ receptors in humans, mice, and rats are FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). These receptors bind to the Fc region of the IgG to help clear immune complexes.

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GENES

100

101

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Ms IgG1, κ-FITC (A112-2)

NKR-P1A-FITC (10/78)

Figure 15

Figure 16

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Figures 13-16. Blocking of aggregated Ms IgG1, κ-FITC (A112-2) and anti-NKR-P1A-FITC (10/78) on rat splenocytes by Rat FcBlock™ (CD32, D34-485). A112-2 and 10/78 lots selected for testing contained 2-4% aggregates and approximately 4% of degraded products. FcR-mediated binding of aggregates to B cells (OX-33 positive cells) was eliminated when cells were pre-incubated with Rat FcBlock™ (Figures 15,16) compared to cells pre-incubated with the isotype control (for Rat FcBlock™) (Figures 13,14).

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T Lymphocytes

T cells can be classified not only based on the expression of CD4 or CD8 and cytokine secretion profiles but also on the specific alpha or beta chain of TCR they bear on their surfaces.

CELLS

PROTEINS

Cell Number

used to study the expression pattern of leukocyte antigens on T cells. Subsets of mature T cells can be delineated by the expression of either CD4 or CD8, and these subsets can be further subdivided according to the types of cytokines the T cells secrete. T cells are important mediators of cytotoxicity and help to coordinate cell mediated and humoral immune responses.

CD25-FITC (0X-39)

CD3-APC (1F4)

CD3-APC (1F4)

Cell Number

T cells, so named because they mature in the thymus, are a class of small lymphocytes that can be distinguished from other hematopoietic cells based on their function and expression of the T cell receptor (TCR). The TCR complex consists of a dimeric hypervariable T cell receptor, which binds antigen, and an associated invariant CD3 complex, which is involved in TCR signal transduction. CD3-specific monoclonal antibodies (mAb) are well suited as markers for mature T cells and in combination with multicolor immunofluorescence staining and flow cytometric analysis, are

GENES

TCR αβ-FITC (R73)

CD134-FITC (OX-40)

Figures 21, 22. Expression of CD25 (IL-2R alpha chain) and CD134 (OX-40) on rat spleen T cells (CD3+) following in vitro incubation for 48 hours with medium alone (red line) or activation for 48 hours with Concanavalin A (green line).

TCR γδ-FITC (V65)

Figure 18

CD3-APC (1F4)

CD3-APC (1F4)

Figure 17

TCR Vα 8-FITC (G177)

Figure 19

TCR Vβ 8.2/8.4-FITC (R78)

Figure 20

Figures 17-20. Total lymph node cells stained simultaneously with anti-CD3-APC (1F4) and FITCconjugated (or biotinylated mAb + SAV-FITC) anti-TCR antibodies (as noted on the x-axis).

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CELLS

CD3-APC (1F4)

GENES PROTEINS

Figure 26

Figure 27 CD4-FITC (OX-35)

CD3-APC (1F4)

CD3-APC (1F4)

CD4 (OX-38) on frozen spleen

Isotype Control-FITC (G155-178)

Figure 23 Figure 28

Figure 29 CD5-FITC (OX-19)

CD3-APC (1F4)

CD5 (OX-19) on frozen spleen

CD2 (OX-34) on frozen spleen Figure 30

Figure 24

Figure 31 CD8a-FITC (OX-8)

CD3-APC (1F4)

CD3-APC (1F4)

CD8a (OX-8) on paraffinembedded spleen

CD2-FITC (OX-34)

Figures 23-35. Flow cytometric data of rat peripheral blood cells stained with the CD3-APC (1F4) to identify T cells and a panel of FITC-conjugated antibodies (or biotinylated mAb + SAV-FITC) that react with cell-surface proteins of interest on T cells. The same antibody (in purified format) was used for immunohistochemical staining of rat tissue sections.

Figure 33

Figure 32 Pan-T Cell Marker-FITC (OX-52)

Pan-T cell marker (OX-52) on frozen lymph node

CD3-APC (1F4)

Figure 25

Figure 35

Figure 34 CD43 (HIS17) on paraffinembedded spleen

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CD43-FITC (HIS17)

NK Cells NK cells are large granular lymphocytes that do not express either a BCR or a TCR and are capable of non-MHC restricted cell mediated cytotoxicity without prior sensitization or coating of the target cell with antibody. NK cells are also the effector cells for antibody dependent cell mediated cytotoxicity, a process

in which cells coated with antibody are destroyed following NK cell degranulation and release of perforin and granzymes. NK cells in the rat can be distinguished either by high levels of NKR-P1A expression or by the fact that the majority of NK cells express CD8a, but do not co-express CD3.

NKR-P1A-PE (10/78)

NKR-P1A-PE (10/78)

GENES CELLS

PROTEINS

NKR-P1A (10/78), frozen spleen Figure 41. IHC staining of rat spleen section using the 10/78 mAb. CD2-FITC (OX-34)

Figure 36

Figure 37

NKR-P1A-PE (10/78)

NKR-P1A-PE (10/78)

Isotype Control-FITC (G155-178)

CD3-FITC (1F4)

CD43-FITC (HIS17)

Figure 38

Figure 39

NKR-P1A-PE (10/78)

Figures 36-40. Rat peripheral blood cells stained with 10/78, a mAb specific for NKR-P1A, and a panel of FITC-conjugated antibodies that react with cell-surface proteins of interest on NK cells.

CD54-FITC (1A29)

Figure 40

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Monocytes/Macrophages

GENES PROTEINS

Blood CD3- CD4+ cells

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Cell Number

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CELLS

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differential light scattering properties of granulocytes and monocytes/lymphocytes (see next section). A second means for identifying rat monocytes is based on the fact that these cells express CD4 but do not coexpress CD3. This scheme allows effective analysis of the monocytic lineage utilizing BD PharMingen’s broad range of antibodies for rat leukocytes. Following the migration of monocytes to a tissue and their subsequent maturation, phenotypic changes are observed on mature macrophages (Figures 42,43).

Spleen CD3- CD4+ cells

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Cell Number

Figure 50

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Figures 44-51. Flow cytometric staining of several cell surface antibodies to rat peripheral blood monocytes characterized by the expression of CD4 (OX-35) and lack of CD3 (1F4).

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Figure 49

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Granulocyte Marker-FITC (HIS48)

Figure 48

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CD61-FITC (F11)

CD80-FITC (3H5)

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Cell Number

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Figure 46

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Cell Number 104

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CD11a-FITC (WT.1)

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Figure 47

Cell Number

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Cell Number

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CD11b-FITC (WT.5)

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Figure 45

Figure 44

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Isotype Control-FITC (G155-178)

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CD4-PE (OX-35)

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Cell Number

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Cell Number

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CD3-APC (1F4)

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Figures 42, 43. Expression of MHC class II RT1B on peripheral blood monocytes (CD3-CD4+) and upregulation on splenic macrophages (CD3-CD4+).

CD3- CD4+ Monocytes

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RT1B-FITC (OX-6)

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CD4+ T cells

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CD4- T cells

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Gated on Monocyte Population

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Cell Number

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RT1B-FITC (OX-6)

Monocytes and macrophages constitute a second major class of leukocytes. Macrophages play an important role in immune response due to their phagocytic properties, their ability to secrete cytokines and chemokines, and their ability to function as antigen presenting cells. Monocytes differ from macrophages in that monocytes are immature circulating precursors to the various types of tissue associated macrophages. Rat blood monocytes can be distinguished from other leukocytes using an antibody such as HIS48 (specific for monocyte and granulocyte lineages) combined with a gating scheme that takes advantage of the

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CD86-FITC (24F)

Figure 51

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Granulocytes

GENES

Granulocytes comprise the last major class of hematopoietic cells to be discussed here. Granulocytes characteristically possess numerous cytoplasmic granules. These cells play a role in inflammatory responses

and are capable of phagocytizing opsonized debris. Granulocytes can best be distinguished from other leukocytes when using flow cytometric analysis by their uniquely high levels of side light scatter.

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Forward Scatter Figure 52

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CD11b-APC (WT.5) Figure 55

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CD11a-APC (WT.1) Figure 54

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Isotype Control-APC (G155-178) Figure 53

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Lymphocytes/ Monocytes

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Granulocytes

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CD61-APC (F11) Figure 56

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Granulocyte Marker-APC (HIS48) Figure 57

Figures 52-57. Flow cytometric analysis of granulocytes characterized by a high side scatter profile stained with a panel of biotinylated anti-rat cell surface antibodies + SAV-APC.

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CELLS

PROTEINS

Cell-Surface Adhesion Molecules

GENES

Adhesion molecules play an important role in several aspects of an immune response. These molecules control the homing and migration of leukocytes and mediate many of the direct intercellular interactions essential for successful immune responses. Families of adhesion molecules involved in immune responses include the integrins, the selectins, and members of the Ig superfamily.

PROTEINS

Cell Number

CELLS

CD11a-FITC (WT.1)

CD11a (WT.1) on frozen spleen Figure 59

Cell Number

Figure 58

CD18-FITC (WT.3)

CD18 (WT.3) on paraffin-embedded spleen

CD29 (Ha2/5) on frozen spleen

Figure 61

CD3-PE (1F4)

Figure 60

Integrins The integrins are a large and diverse family of cell surface molecules that mediate interactions of cells with components of the extracellular matrix, as well as interactions between cells. Integrins are noncovalently linked heterodimers consisting of a large α chain (for example CD11a, b, c and CD49a, b, d, c) paired with a smaller β chain (for example CD18, CD29, and CD61). Much of the diversity within the integrin family is generated by the association of different α and β integrins to form unique integrin heterodimers. These molecules play an important role in inflammatory and immune responses (Figures 58-63).

Selectins The selectins are a family of carbohydrate binding cell adhesion molecules that play an important role in leukocyte homing. L-selectin (CD62L) is expressed on nearly all circulating leukocytes and is involved in leukocyte homing via interactions with vascular addressins. P-selectin (CD62P) is expressed on activated platelets and endothelial cells where it mediates the adhesion of neutrophils and monocytes to the activated cells. E-selectin (CD62E) is expressed on activated endothelium where it also plays a role in the migration of immune cells (Figure 64). Ig Superfamily A third family of adhesion molecules are those which are members of the immunoglobulin (Ig) superfamily. The Ig superfamily includes over 100 members many of which are adhesion molecules (for example CD2, CD31, CD54, and CD106) (Figures 65-66 on page 11).

CD3-PE (1F4)

CD29-FITC (Ha2/5)

CD29 (Ha2/5) on frozen spleen Figure 62

Figure 63

Figures 58-63. Flow cytometric and IHC data generated from rat spleen using antibodies specific for the indicated members of the integrin family. CD62L-FITC (HRL1)

Figure 64. Flow cytometic analysis of rat spleen cells stained simultaneously with CD3-PE (1F4) and CD62L-FITC (HRL1)

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Rat MHC Haplotype Chart

GENES

RT1 Haplotype

Strains

a av1 b c d dv1 e f g h i j k l lvl m n o p q sa sb u

AVN DA, ACI, ACP, COP BUF, ALB AUG, PVG BVD BDIX, TAL BDVII AS2 KGH HW BI LEJ WKA/Hok, WKAH, KYN, OKA, SHR LEW F344 MNR BN, MAXX MR RP NIG III NSD WiN WF, TO, AO, LOU, WAG, SDJ, YO, BB/W, DZB

CELLS

PROTEINS

MAdCAM-1 (OST20) on paraffinembedded spleen Figure 65

Specificity

Clone

Isotype

Format

Size

Cat. No.

CD2 (LFA-2)

OX- 34

Mouse IgG2a

CD3

G4.18

Mouse IgG3

Purified FITC PE NA/LE™ Purified Biotin FITC PE Purified FITC PE APC Purified Biotin FITC PE Cy-Chrome™ APC Purified FITC PE Purified FITC PE Purified

0.5 0.5 0.1 0.5 0.5 0.5 0.5 0.2 0.5 0.5 0.1 0.1 0.5 0.5 0.5 0.2 0.1 0.1 0.5 0.5 0.2 0.5 0.5 0.2 0.5

mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg

22001D 22004D 22005A 22010D 22011D 22012D 22014D 22015B 22731D 22734D 22735A 22739A 22021D 22022D 22024D 22025B 22028A 22029A 22031D 22034D 22035B 22051D 22054D 22055B 22061D

Purified Biotin FITC PE Purified FITC Purified Biotin FITC

0.5 0.5 0.5 0.2 0.1 0.1 0.5 0.5 0.5

mg mg mg mg mg mg mg mg mg

22071D 22072D 22074D 22075B 22421A 22424A 22501D 22502D 22504D

CD3

1F4

Mouse IgM

CD4

OX-35

Mouse IgG2a

CD4

OX- 38

Mouse IgG2a

CD5

OX-19

Mouse IgG1

CD5

HIS47

CD8a

OX-8

Mouse IgG2a Mouse IgG1

CD8a

G28

Mouse IgG2a

CD8b

341

Mouse IgG1

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CD3-APC (1F4)

Antibodies to Rat Leukocytes and Related Cells

CD54-FITC (1A29)

Figure 66

Figures 65, 66. Flow cytometric and IHC data generated from rat spleen using antibodies specific for adhesion molecules belonging to the Ig superfamily.

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GENES CELLS

PROTEINS

Antibodies to Rat Leukocytes and Related Cells, continued Specificity

Clone

Isotype

Format

Size

Cat. No.

CD9

RPM.7

Mouse IgG3

Purified FITC Purified FITC PE Purified Biotin FITC NA/LE™ Purified Biotin FITC PE Purified

0.5 0.5 0.1 0.5 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.2 0.1

mg mg mg mg mg mg mg mg mg mg mg mg mg mg

22791D 22794D 22541A 22544D 22545B 22531D 22532D 22534D 22080D 22081D 22082D 22084D 22085B 33601A

NA/LE™ Purified FITC Purified Biotin FITC PE Purified FITC PE NA/LE™ Purified Biotin PE NA/LE™ Purified Biotin FITC PE NA/LE™

0.5 0.5 0.5 0.1 0.1 0.5 0.2 0.1 0.1 0.1 0.5 0.1 0.1 0.1 0.5 0.1 0.1 0.5 0.1 0.5

mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg

22520D 22521D 22524D 22091A 22092A 22094D 22095B 22811A 22814A 22815A 09630D 09631A 09632A 09635A 22600D 22601A 22602A 22604D 22605A 22590D

NA/LE™ Purified Biotin FITC Purified

0.5 0.5 0.5 0.5 0.5

mg mg mg mg mg

22630D 22631D 22632D 22634D 09871D

NA/LE™ Purified Biotin PE NA/LE™ Purified Purified FITC Purified Biotin Purified FITC Purified Biotin Purified FITC PE Purified FITC Purified Biotin FITC PE Cy-Chrome™

0.5 0.5 0.5 0.1 0.5 0.5 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.2 0.1 0.1 0.5 0.5 0.5 0.2 0.1

mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg

22710D 22711D 22712D 22715A 22880D 22881D 22881A 22884D 22821D 22822D 22771D 22774D 22101D 22102D 22111D 22114D 22115B 22121A 22124A 22131D 22132D 22134D 22135B 22138A

CD11a WT.1 (Integrin αL chain LFA-1α chain) CD11b WT.5 (Integrin aM chain, Mac-1 α chain) CD11b/c OX-42

CD15s 2H5 [Sialyl Lewis X (SLex)] CD18 WT.3 (Integrin β2 chain)

Mouse IgG2a

Mouse IgA

Mouse IgG2a

Mouse IgM Mouse IgG1

CD25 (IL-2R α chain)

OX - 39

Mouse IgG1

New CD26

OX-61

Mouse IgG2a

New CD27

LG.3A10

Hamster IgG1

CD28

JJ319

Mouse IgG1

CD28

JJ316

Mouse IgG1 Armenian Hamster IgM

CD29 Ha2/5 (Integrin β1 chain)

CD29 HMβ1-1 (Integrin β1 chain) CD31 (PECAM-1)

TLD-3A12

Armenian Hamster IgG, Group 2 Mouse IgG1

New CD32

D34-485

Mouse IgG1

New OX-40 Ligand CD42d

ATM-2

Mouse IgG1

RPM.4

Mouse IgG2a

HIS17

Mouse IgG1

OX-49

Mouse IgG2a

CD43 (Leukosialin) CD44 H (Pgp-1, H-CAM)

CD45.2 HIS41 Mouse IgG1 (RT7.2 of Leukocyte Common Antigen) CD45 OX-1 Mouse IgG1 (Leukocyte Common Antigen)

12

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Antibodies to Rat Leukocytes and Related Cells, continued

GENES

Specificity

Clone

Isotype

Format

Size

Cat. No.

CD45R

HIS24

Mouse IgG2b

CD45RA

OX-33

Mouse IgG1

CD45RC

OX-22

Mouse IgG1

CD45RC

HIS25

Mouse IgG1

Purified FITC PE Purified Biotin FITC PE Cy-Chrome™ Purified FITC PE Purified

0.5 0.5 0.2 0.5 0.5 0.5 0.2 0.1 0.5 0.5 0.2 0.5

mg mg mg mg mg mg mg mg mg mg mg mg

22161D 22164D 22165B 22171D 22172D 22174D 22175B 22178A 22181D 22184D 22185B 22221D

NA/LE™ Purified

0.5 mg 0.5 mg

22620D 22621D

Purified FITC

0.5 mg 0.5 mg

22611D 22614D

Purified FITC Purified

0.5 mg 0.1 mg 0.5 mg

01871D 01874A 01881D

Purified FITC NA/LE™ Purified Biotin FITC PE Purified FITC NA/LE™ Purified FITC NA/LE™ Purified Biotin FITC PE Purified Biotin FITC PE Purified

0.5 0.5 0.5 0.5 0.5 0.5 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.2 0.1 0.1 0.5 0.2 0.1

mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg

22751D 22754D 22490D 22491D 22492D 22494D 22495B 22721D 22724D 22440D 22441D 22444D 01860D 01861D 01862D 01864D 01865B 22471A 22472A 22474D 22475B 22481A

Purified

0.1 mg

09361A

Purified FITC PE NA/LE™ Purified Biotin PE Purified

0.5 0.5 0.2 0.5 0.5 0.5 0.2 0.5

mg mg mg mg mg mg mg mg

22191D 22194D 22195B 22660D 22661D 22662D 22665B 22671D

Purified Biotin FITC Purified Biotin FITC PE PerCP

0.5 0.5 0.5 0.5 0.5 0.5 0.2 0.1

mg mg mg mg mg mg mg mg

22201D 22202D 22204D 22211D 22212D 22214D 22215B 2221PA

CD49a Ha31/8 (Integrin α1 chain) CD49b Ha1/29 (Integrin α2 chain) CD49d MRα4-1 (Integrin α4 chain) CD49e HMα5-1 (Integrin α5 chain)

Armenian Hamster IgG, Group 2 Armenian Hamster IgG, Group 2 Mouse IgG2a

CD53

OX-44

Armenian Hamster IgG, Group 1 Mouse IgG1

CD54 (ICAM-1)

1A29

Mouse IgG1

CD59

TH9

Mouse IgG1

CD61 F11 (Integrin β3 chain)

Mouse IgG1

CD61 2C9.G2 (Integrin β3 chain)

Armenian Hamster IgG, Group 1

CD62L HRL1 (L-selectin, LECAM-1)

Armenian Hamster IgG, Group 2

CD62L HRL2 (L-selectin, LECAM-1)

Armenian Hamster IgG, Group 1 Rabbit IgG

CD62P Polyclonal (P-selectin) CD71 OX-26 (Transferrin Receptor) CD80 (B7-1)

3H5

Mouse IgG2a

Mouse IgG1

CD86 (B7-2)

24F

Mouse IgG1

CD90 (Thy-1)

HIS51

Mouse IgG2a

CD90 (Thy-1)

OX-7

Mouse IgG1

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CELLS

13

PROTEINS

GENES CELLS

PROTEINS

Antibodies to Rat Leukocytes and Related Cells, continued Specificity

Clone

Isotype

Format

Size

Cat. No.

CD122 (IL-2Rβ chain)

L316

Mouse IgG1

CD134 (OX-40 Antigen)

OX-40

Mouse IgG2b

β2 Microglobulin

TLD-3H12b

Mouse IgG1

Crry/p65

512

Mouse IgG1

Purified Biotin FITC Purified Biotin FITC Purified FITC Purified

0.5 0.5 0.5 0.1 0.5 0.5 0.5 0.5 0.5

mg mg mg mg mg mg mg mg mg

22741D 22742D 22744D 22041A 22042D 22044D 22801D 22804D 22581D

Dendritic Cells Fas Ligand

OX-62 MFL4

Granulocytes

HIS48

Mouse IgG1 Armenian Hamster IgG, Group 3 Mouse IgM

Purified NA/LE™ Purified Biotin Purified Biotin FITC Purified Biotin PE NA/LE™ Purified Purified

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.1 0.5 0.5 0.5

mg mg mg mg mg mg mg mg mg mg mg mg mg

22651D 22700D 22701D 22702D 22261D 22262D 22264D 22851D 22852D 22855A 22840D 22841D 22691D

Purified PE NA/LE™ Purified Purified Biotin FITC Purified Biotin Purified Biotin FITC PE Purified FITC Purified

0.5 0.2 0.5 0.1 0.1 0.1 0.5 0.5 0.1 0.5 0.5 0.5 0.2 0.5 0.5 0.5

mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg

22231D 22235B 22860D 22861A 22831A 22832A 22834D 22451D 22452A 22641D 22642D 22644D 22645B 22251D 22254D 22241D

Purified PE

0.5 mg 0.1 mg

22681D 22685A

New Granulocytes RP-1

Mouse IgG2a

New Granulocytes RP-3

Mouse IgM

Macrophage Activator Macrophage Subset New MAdCAM-1

anti-RMA

Mouse IgG1

HIS36

Mouse IgG2a

OST2

Mouse IgG1

HIS57

Mouse IgG1

Mononuclear 1C7 Phagocyte NKR-P1A (CD161) 10/78

Mouse IgG1

Pan-T Cells

OX-52

Mouse IgG2a

QCA-1

HIS45

New VCAM-1 (CD106)

MR106

Mouse IgG1 Mouse IgG1

New Marginal Zone B Cells

Mouse IgG1

Antibodies to Rat Major Histocompatability Complex (MHC) Antigens Specificity

Clone

Isotype

Format

Size

Cat. No.

RT1A

OX-18

Mouse IgG1

RT1A a, b

C3

Rat IgG2b

RT1B a, b, l

B5

Rat IgM

RT1B

OX-6

Mouse IgG1

Purified Biotin FITC PE Purified Biotin FITC Purified FITC NA/LE™ Purified Biotin FITC PE PerCP Purified

0.5 0.5 0.5 0.1 0.5 0.1 0.5 0.1 0.1 0.5 0.5 0.5 0.5 0.2 0.1 0.5

mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg

22301D 22302D 22304D 22305A 22351D 22352A 22354D 22371A 22374A 22380D 22331D 22332D 22334D 22335B 2233PA 22311D

Purified FITC

0.5 mg 0.5 mg

22341D 22344D

RT1B HIS19 (non-n haplotypes) RT1D OX-17

14

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Mouse IgG1 Mouse IgG1

Antibodies to Rat T-Cell Receptors (TCR)

GENES

Specificity

Clone

Isotype

Format

Size

Cat. No.

αβ TCR

R73

Mouse IgG1

γδ TCR

V65

Mouse IgG1

γδ TCR Subset

V45

Mouse IgG1

Vα 4 TCR

G99

Mouse IgG1

NA/LE™ Purified Biotin FITC PE PerCP Purified Biotin FITC PE Purified Biotin Purified

0.5 0.5 0.5 0.5 0.2 0.1 0.5 0.1 0.5 0.2 0.5 0.5 0.5

mg mg mg mg mg mg mg mg mg mg mg mg mg

22280D 22281D 22282D 22284D 22285B 2228PA 22461D 22462A 22464D 22465B 22511D 22512D 22561D

Vα 8 TCR

G177

Mouse IgG1

Purified

0.5 mg

22551D

Vβ 8.21 and Vβ 8.4a TCR Vβ 8.5 TCR

R78

Mouse IgG1

B73

Mouse IgG1

Purified FITC Purified

0.5 mg 0.1 mg 0.5 mg

22391D 22394A 22401D

Vβ 10 TCR

G101

Mouse IgG2a

FITC

0.5 mg

22414D

Vβ 16 TCR

HIS42

Mouse IgG2b

Purified

0.5 mg

22291D

CELLS

Isotype Controls Specificity

Clone

Format

Size

Mouse IgG1, κ Mouse IgG1, κ (anti-KLH)

MOPC-31C

Purified

0.5 mg

Cat. No. 03251D

A112-2

Mouse IgG1, κ (b allotype, anti-KLH) Mouse IgG1, λ (anti-TNP) Mouse IgG1, λ

A115-2

NA/LE™ Purified Biotin FITC PE Cy-Chrome™ PerCP Purified

0.5 mg 0.5 mg 0.25 mg 0.25 mg 0.1 mg 0.1 mg 0.1 mg 0.5 mg

03210D 03211D 03212C 03214C 03215A 03218A 0321PA 03221D

A111-3

Purified

0.5 mg

03191D

S1-68.1

Purified

0.5 mg

03011D

Mouse IgG2a, κ (anti-TNP)

G155-178

NA/LE™ Purified Biotin FITC PE Cy-Chrome™ Purified

0.5 mg 0.5 mg 0.25 mg 0.25 mg 0.1 mg 0.1 mg 0.5 mg

03020D 03021D 03022C 03024C 03025A 03028A 03031D

0.25 mg 0.25 mg 0.1 mg 0.5 mg

03042C 03044C 03045A 03051D

Mouse IgG2a, λ

HOPC-1

Mouse IgG2b, κ (anti-TNP)

49.2

Mouse IgG2b, λ Mouse IgG3, κ (anti-Fructosan) Mouse IgG3, κ (anti-KLH)

G11-59

Biotin FITC PE Purified

J606

NA/LE™

0.5 mg

03060D

A112-3

Mouse IgG3, λ

G19-143

Purified Biotin FITC PE Purified

0.5 mg 0.25 mg 0.25 mg 0.1 mg 0.5 mg

03201D 03202C 03204C 03205A 03071D

Mouse IgM, κ (anti-TNP)

G155-228

Mouse IgM, λ (anti-Dextran) Mouse IgA, κ

MOPC-104E

Purified Biotin FITC Purified

0.5 mg 0.25 mg 0.25 mg 0.5 mg

03081D 03082C 03084C 03091D

Purified Biotin FITC

0.5 mg 0.25 mg 0.25 mg

03101D 03102C 03104C

M18-254

www.bdbiosciences.com

15

PROTEINS

GENES CELLS

PROTEINS

Isotype Controls, continued Specificity

Clone

Format

Size

Cat. No.

Mouse IgA, λ Mouse IgE, κ (anti-dansyl) Mouse IgE, κ (anti-TNP) Mouse IgE, κ (b allotype, anti-TNP) Hamster IgG, κ group 1, (anti-TNP)

MOPC-315 27-74

Purified Purified

0.5 mg 0.5 mg

03111D 03131D

C38-2

Purified

0.5 mg

03231D

C48-2

Purified

0.5 mg

03241D

A19-3

Hamster IgG, λ group 1, (anti-TNP)

G235-2356

Hamster IgG, λ group 2, (anti-KLH)

Ha4/8

Hamster IgG, λ group 3, (anti-TNP)

A19-4

Hamster IgM, λ (anti-TNP)

G235-1

NA/LE™ Purified Biotin FITC PE Cy-Chrome™ APC PerCP NA/LE™ Purified Biotin FITC PE Cy-Chrome™ NA/LE™ Purified Biotin FITC PE Cy-Chrome™ APC NA/LE™ Purified Biotin FITC PE NA/LE™ Purified Biotin FITC

0.5 mg 0.5 mg 0.25 mg 0.25 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.5 mg 0.5 mg 0.25 mg 0.25 mg 0.1 mg 0.1 mg 0.5 mg 0.5 mg 0.25 mg 0.25 mg 0.1 mg 0.1 mg 0.1 mg 0.5 mg 0.5 mg 0.25 mg 0.25 mg 0.1 mg 0.5 mg 0.5 mg 0.25 mg 0.25 mg

11150D 11151D 11152C 11154C 11155A 11158A 11159A 1115PA 11120D 11121D 11122C 11124C 11125A 11128A 11140D 11141D 11142C 11144C 11145A 11148A 11149A 11160D 11161D 11162C 11164C 11165A 11130D 11131D 11132C 11134C

16

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Cytokines

Detection of Intracellular Cytokines by Flow Cytometry

Isotype Control-PE (A112-2)

Figure 67

11.8%

CD4-FITC (OX-38)

Figure 68

10.7%

CD4-FITC (OX-38)

Figure 69

required for making statistically significant measurements. In addition to fluorochromeconjugated antibodies for intracellular staining of rat cytokines, BD PharMingen provides Cytofix/Cytoperm Intracellular flow cytometry kits that provide fixation and permeabilization buffers and protein transport inhibitors. Also now available are RiCK (Rat intracellular CytoKine) positive control cells. These cell suspensions contain fixed, nonpermeabilized rat lymphoid cells that express easily detected levels of intracellular cytokines and can thus be used as positive controls for experiments.

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CD4-FITC (OX-38)

17

GM-CSF-PE (B61-5)

Multicolor immunofluorescent staining with antibodies against intracellular cytokines and cell surface markers provides a highresolution method to determine the nature and frequency of cells which express a particular cytokine(s). For example, staining of an individual cell surface antigen and two cytoplasmic cytokines has been used to identify and enumerate cell types that express cytokines in either a polarized (e.g., Th1- versus Th2-like cells) or unpolarized (e.g., Th0-like cells) pattern. In addition to enabling highly specific and sensitive measurements of several parameters for individual cells simultaneously, this method has the capacity for rapid analysis of large numbers of cells, which are

PROTEINS

IL-4-PE (OX-81)

BD PharMingen offers a growing line of reagents for the study of rat cytokines and chemokines including recombinant rat cytokines, specific antibodies to rat cytokines and chemokines, and optimized immunoassays. These antibodies are offered in formats useful for detection of cytokine producing cells by flow cytometry, determination of cytokine protein levels by sandwich ELISA, as well as funcional analysis utilizing biologically active recombinant proteins, and neutralizing anti-cytokine antibodies.

CELLS

IL-10-PE (A5-4)

the experimental system. BD PharMingen has designed its line of cytokine reagents in response to the needs of researchers for simultaneous analysis of multiple cytokines.

13.6%

CD4-FITC (OX-38)

Figure 70

Side light scatter

The development and function of the immune system depends on the production and response to cytokines. Cytokines are pleiotropic mediators capable of stimulating multiple biological effects. Cytokines have taken center stage in many clinical and basic research studies because they play such a primary role in transmitting regulatory signals between various types of cells. The intercellular communication system consisting of cytokines and their receptors is comprised of multiple stimulatory and inhibitory cytokines. The complexity of the cytokine network often necessitates a multi-parameter approach to study the functional roles played by a particular cytokine, since each cytokine’s role may be influenced by other cytokines which are present in

GENES

Forward light scatter

Figure 71 Figures 67-71. RiCK-2 positive control cells stained with FITCconjugated mouse anti-rat CD4 (OX-38), washed, permeabilized, and subsequently stained with PE mouse IgG1 isotype control (A112-2), PE-mouse anti-rat IL-4 antibody (OX-81), PE-mouse anti-rat IL-10 (A5-4) or PE mouse anti-rat GM-CSF (B61-5). Figure 74. The lightscattering characteristics for RiCK-2 cells are shown.

GENES

Antibodies for Inracellular Cytokine and Chemokine Flow Cytometry Antibodies for Rat Cytokines

CELLS

PROTEINS

Specificity Clone

Isotype

Format

Application

Size

Cat. No.

IL-4

OX-81

Mouse IgG1

IFN-γ

DB-1

Mouse IgG1

IL-10

A5-4

Mouse IgG2b

TNF-α

TN3-19.12 Hamster IgG

GM-CSF

B61-5

Purified PE Purified FITC PE Purified PE Purified PE Purified PE

IC/Flow IC/Flow IC/Flow IC/Flow IC/Flow IC/Flow IC/Flow IC/Flow IC/Flow IC/Flow IC/Flow

0.1 mg 0.1 mg 50 ng 100 tests 100 tests 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg

24051A 24054A 20441N 20444X 20445X 24091A 24095A 23351A 23355A 24131A 24135A

Purified PE

IC/Flow blocking 0.1 mg IC/Flow staining 0.1 mg

Mouse IgG1

Antibodies for Rat Chemokines MCP-1 2H5 Hamster IgG

blocking staining blocking staining staining blocking staining blocking staining blocking staining

18241A 18245A

Cell Fixation and Permeabilization Kits and Components Cytofix/Cytoperm™ Kit Cytofix/Cytoperm™ Kit with GolgiStop™ Cytofix/Cytoperm™ Kit with GolgiPlug™ GolgiStop™ (with Monensin) GolgiPlug™ (with Brefeldin A) Cytofix/Cytoperm™ Buffer Perm/Wash™ Buffer NEW BrdU Flow Kit

250 tests 250 tests 250 tests 1.0 ml 0.7 ml 100 mls 100 mls 50 tests

Rat Intracellular Cytokine Positive Control Cells RiCK-2 (Positive for IL-4, IL-10, GM-CSF, IFN-γ, TNF-α)

5x10 cells 24142Z

Antibodies for Rat Cytokine Receptors IL-2Rα OX-39 Mouse IgG1 Purified Biotin FITC PE IL-2Rβ L316 Mouse IgG1 Purified Biotin FITC

0.5 0.5 0.5 0.2 0.5 0.5 0.5

Flow Cytometry

Flow Cytometry

2075KK 2076KK 2300KK 2092KZ 2301KZ 2090KZ 2091KZ 2354KK

6

mg mg mg mg mg mg mg

Reagents for Immunofluoresent Staining of Cell Suspensions PharMingenStain Buffer (with FBS) PharMingenStain Buffer (with BSA) Cytofix™ Buffer

22091D 22092D 22094D 22095B 22741D 22742D 22744D

20151E 20161E 2014KZ

Neutralizing Rat Cytokine Antibodies IL-2 Polyclonal Rabbit Ig IL-4 OX-81 Mouse IgG1

NA/LE™ NA/LE™

Neutralization Neutralization

0.5 mg 0.5 mg

24030D 24050D

TNF-α

NA/LE™

Neutralization

0.5 mg

23350C

Bioactive/ELISA Bioactive/ELISA Bioactive/ELISA ELISA only Bioactive/ELISA Bioactive Bioactive/ELISA Bioactive/ELISA

5 5 5 5 5 2 5 5

25001V 25011V 25081V 25071V 25051V 25061V 25031V 25041V

TN3-19.12 Hamster Ig

Recombinant Rat Cytokines and Chemokines IL-2 IL-4 IL-6 IL-10 (ELISA Standard) GM-CSF IFN-γ TNF-α MCP-1

18

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µg µg µg µg µg µg µg µg

BD OptEIA™ Rat Cytokine ELISA Sets BD OptEIA™ ELISA Sets are specially formulated to facilitate accurate measurement of soluble proteins in tissue culture supernatents, sera, plasma, and other complex biological fluids. Each set consists of pre-titered capture and detection antibodies along with lyophilized recombinant cytokine standard and horseradish peroxidase conjugate sufficient for 20

GENES

96-well plates. The BD OptEIA™ Set reagents are specifically formulated to reduce background and non-specific binding caused by autoantibodies, rheumatoid factor, and plasma proteins such as complement, fibronectin and Fc receptors. Typical standard curves from BD OptEIA™ Sets for rat IL-2, IL-4, IL-10, IFN-γ, TNF-α, and MCP-1 are shown below.

US Price 550 Rat IL-2 BD OptEIA™ Set

Contents: • Capture Antibody: Anti-rat IL-2 • Detection Antibody: Biotinylated anti-rat IL-2 • Enzyme Reagent: HRP-conjugated avidin • Standard: Recombinant rat IL-2 (lyophilized) • Standard Curve Range: 31.3 - 2000 pg/ml

Optical Density (450 nm)

Rat IL-2 OptEIA™ Set; Cat. No. 2628KI 1

0.1

0.01 10

Optical Density (450 nm)

1

0.1

0.01 1

Optical Density (450 nm)

1

0.1

0.01 10

Optical Density (450 nm)

Rat IFN-γ BD OptEIA™ Set

IFN-γ (pg/ml)

Optical Density (450 nm)

Rat TNF-α BD OptEIA™ Set

TNF-α (pg/ml)

Rat MCP-1 OptEIA™ Set; Cat. No. 2610KI

Rat MCP-1 BD OptEIA™ Set Optical Density (450 nm)

Contents: • Capture Antibody: Anti-rat MCP-1 • Detection Antibody: Biotinylated anti-rat MCP-1 • Enzyme Reagent: HRP-conjugated avidin • Standard: Recombinant rat MCP-1 (lyophilized) • Standard Curve Range: 31.3 - 2000 pg/ml

1000

100

IL-10 (pg/ml)

Rat TNF-α OptEIA™ Set; Cat. No. 2697KI Contents: • Capture Antibody: Anti-rat TNF-α • Detection Antibody: Biotinylated anti-rat TNF-α • Enzyme Reagent: HRP-conjugated avidin • Standard: Recombinant rat TNF-α (lyophilized) • Standard Curve Range: 31.3 - 2000 pg/ml

100

Rat IL -10 BD OptEIA™ Set

Rat IFN-γ OptEIA™ Set; Cat. No. 2696KI Contents: • Capture Antibody: Anti-rat IFN-γ • Detection Antibody: Biotinylated anti-rat IFN-γ • Enzyme Reagent: HRP-conjugated avidin • Standard: Recombinant rat IFN-γ (lyophilized) • Standard Curve Range: 31.3 - 2000 pg/ml

10

IL-4 (pg/ml)

Rat IL-10 OptEIA™ Set; Cat. No. 2611KI Contents: • Capture Antibody: Anti-rat IL-10 • Detection Antibody: Biotinylated anti-rat IL-10 • Enzyme Reagent: HRP-conjugated avidin • Standard: Recombinant rat IL-10 (lyophilized) • Standard Curve Range: 15.6 - 1000 pg/ml

1000

Rat IL-4 BD OptEIA™ Set

Rat IL-4 OptEIA™ Set; Cat. No. 2631KI Contents: • Capture Antibody: Anti-rat IL-4 • Detection Antibody: Biotinylated anti-rat • Enzyme regent: HRP-conjugated avidin • Standard: Recombinant rat IL-4 (lyophilized) • Standard Curve Range: 1.6 - 100 pg/ml

100

IL-2 (pg/ml)

1

0.1

0.01 10

100

1000

MCP-1 (pg/ml)

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19

CELLS

PROTEINS

GENES CELLS

PROTEINS

OptEIA™ Sets General ELISA Reagents TMB Substrate Reagent Set; Cat. No. 2642KK

Assay Diluent; Cat. No. 26411E

Contents: • Substrate Reagent A, 300 ml • Substrate Reagent B, 300 ml

Volume: 500 ml

Substrate Reagent A contains hydrogen peroxide in a buffered solution. Substrate Reagent B contains 3, 3’, 5, 5’-tetramethylbenzidine in organic solvent. When mixed together, TMB substrate solution reacts with peroxidaselabeled conjugates to develop a blue color. After addition of stopping solution (1M phosphoric or 2N sulfuric acid), the color changes from blue to yellow and the microwells are read at 450 nm.

Assay Diluent contains animal serum in a buffered solution with 0.15% ProClin-150 as preservative. Intended uses are to block ELISA plates, to dilute the recombinant Standards and samples, and to prepare Working Detector for the BD OptEIA™ Sets ELISA Reagents.

Matched Antibody ELISA Pairs and Protein Standards BD Pharmingen’s extensive portfolio of matched antibody ELISA pairs and protein standards for rat cytokines and chemokines offer the researcher:

• Flexible assay development • Lower cost of ELISA analysis per sample • Reagent use for other applications such as western blotting

Matched Antibody Pairs and Protein Standards for Rat Cytokine and Chemokine ELISA Specificity Description Clone

Isotype

Format

Size

Cat. No.

IL-2

Rabbit Ig Mouse IgG1

Purified Biotin Recombinant Purified Biotin Recombinant Purified Biotin Recombinant Purified Biotin Recombinant Purified Biotin Recombinant Purified Biotin Recombinant Purified Biotin Recombinant

0.5 mg 0.5 mg 5 µg 0.5 mg 0.5 mg 0.5 µg 0.5 mg 0.5 mg 0.5 µg 0.5 mg 0.5 mg 0.5 µg 0.5 mg 0.5 mg 0.5 µg 0.5 mg 0.5 mg 5 µg 0.5 mg 0.5 mg 5 µg

24031D 24042D 25001V 24051D 24112D 25011V 24051D 24112D 25011V 24061D 24072D 25071V 24181D 24172D 25051V 24011D 24022D 24041V 23351D 23442D 25031V

IL-4

IL-4

IL-10

GM-CSF

MCP-1

TNF-α

20

Capture Detection Standard Capture Detection Standard Capture Detection Standard Capture Detection Standard Capture Detection Standard Capture Detection Standard Capture Detection Standard

Polyclonal A38-3 Rec. IL-2 OX-81 Polyclonal Rec. IL-4 OX-81 B11-3 Rec. IL-4 A5-7 A5-5 Rec. IL-10 B61-5/B61-9 B61-10 Rec. GM-CSF C4 B4 Rec.MCP-1 TN3-19.12 Polyclonal Rec.TNF-α

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Mouse IgG1 Rabbit Ig Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Hamster Ig Rabbit Ig

Cell Biology: Apoptosis and Neurobiology

55 p45 Caspase-1

36 31

p33

21 14 1 2 3 4 5

kD 116 97 66 55

Caspase-2

36 31

1

2

3

Rat -

ppRb & pRb

Figure 73. Western blot analysis of caspase-2. Jurkat T cell (lane 1), mouse myeloblasts (lane 2) and DC-3 SV40-transformed, rat ovarian granulosa cells (lane 3) probed with anti-caspase-2 (clone G310-1248, Cat. No. 13951A). The G3101248 antibody identifies caspase-2 as an ~48 kD band.

REF

+

F111

-

+

Figure 74. Western blot analysis of Rb, clone G3245 (Cat. No. 14001A) in cell cycling (+) and quiescent (-) rat cell cultures. Most highly phosphorylated species of Rb are seen in cycling compared to quiescent cultures. REF, primary rat embryo fibroblasts. F111, established rat fibroblasts. ppRb and pRb represent different phosphorylation states of Rb. The higher molecular weight bands (116 kD) are more highly phosphorylated than the lower bands (110 kD).

www.bdbiosciences.com

21

200

101

10 2

103

104

p53-PE (G59-12)

100 60

80

Rat Thymocytes

20

40

kD 116 97 66 55

0 Rat Thymocytes

66

120 160

100

DC3

21

Figure 72. Western blot analysis of caspase-1. HeLa human cervical carcinoma (lane 1), 293 adenovirus-transformed human embryonic kidney (lane 2), Jurkat T cell (lane 3), BALB/c thymocytes (lane 4) and DC-3 SV40-transformed, rat ovarian granulosa cells (lane 5) probed with anti-caspase-1 (clone B24-2, Cat. No. 66441A). Caspase-1 is identified as 45 kD (precursor) and 33 kD (intermediate) bands. In rat species, clone B24-2 primarily detects the 45 kD form of the protein.

80

Cell Number

21

36 31

kD 116 97

DC3

0

36 31

PROTEINS

20

kD 116 97 66 55 p53

Cell Number

Neurobiology In addition to our apoptosis line of products, we have a number of antibodies for the study of cellular and neurobiological systems. BD PharMingen offers a wide variety of antibodies and other reagents for neurobiological research in the rat, including antibodies to neurotransmitters and their receptors, glutamate receptors and structural proteins, tumor suppressors, oncogenes, cell cyclerelated proteins, and a wide variety of other intracellular proteins.

IgG 1 G5 9-1 PA 2 b1 22 PA b2 40

CELLS

IgG 1 G5 9-1 PA 2 b1 22 PA b2 40

Apoptosis Apoptosis, a form of programmed cell death, is a normal physiological process which occurs during normal embryonic development and in the maintenance of tissue homeostasis. Inappropriate induction of apoptosis has broad ranging pathologic implications and as a result, the apoptosis field is of increasing scientific interest, with thousands of new publications occurring every year. BD PharMingen offers a number of reagents designed to identify, quantitate, and characterize apoptosis.

GENES

100

101

10 2

103

10 4

p53-PE (G59-12)

Figure 75. Analysis of p53 expression in SV40transformed rat ovarian cells (DC3) and rat thymocytes. Cells were analyzed by flow cytometry using a p53-PE antibody (G59-12) (green histogram) or an IgG1-PE isotype control (MOPC-21) (open histogram), or by western blot analysis using a panel of p53 antibodies [G59-12 (Cat. no. 14211A), PAb 122 (Cat. no. 14091A), or PAb 240 (Cat. No. 14461A)] or an IgG1 isotype control (MOPC-21, Cat. No. 03171D). The results show that p53 was detected in DC3 cells but not in rat thymocytes. The PEconjugated p53 and IgG1 antibodies are sold as a set (Cat. No. 6733KK).

Cell Biology: Apoptosis and Neurobiology, continued Figure 76. Acetone-fixed, frozen tissue section of rat brain stained for somatostatin (clone YC7,Cat. No. 60132A).

Figure 77. Paraformeldahyde-perfused, frozen rat brain stained with anti-TrkA (Cat. No. 68266E).

Figure 78. Frozen rat brain tissue stained for 5-HT2AR (clone G186-1117, Cat. No. 60231A).

Figure 79. Western blot analysis of 5-HT2AR in rat brain synaptic mambranes. Blots were incubated with either G1861117. (lane 1), G186-1117, absorbed with recombinant GST-5-HT2AR fusion protein (lane 2), or G186-1117 absorbed with an irrelevant (GST-p16) fusion protein (lane 3). The antibody recognizes 2-HT2AR as an ~55 kD band.

5-HT2AR

1

2

3

Figure 80. Frozen rat brain tissue (hippocampus) stained for 5-HT2BR (clone A72-1, Cat. No. 60531A).

kD 200 116 97 66 55

5-HT2CR

Figure 81. Western blot analysis of 5-HT2CR in rat brain synaptic plasma membrane. (Lane 1), clone A4-2 (Cat. No. 60541A) an IgG1 isotype control. (Lane 2), the antibody recognizes 5-HT2CR as e 55kDa band.

36 31 21 14

1

IP:

p5 3 (G SV 59 40 -12 (P ) Ab 10

Figure 82. Formalin-fixed frozen adult rat brain sections (cerebellum) stained with the anti GABAb receptor (Cat. No. 60696E). Arrow indicates GABAb Receptor immunoreactivity. Strong immunostaining is seen in the cell bodies and dendrites of Purkinje cells. B is a higher magnification of A.

Ig G

A

kD 200 116 97 66 55 36 31

B

21

WB: SV40 (PAb 101)

22

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2

0)

1

SV40 Large T Heavy chain

Light chain

Figure 83. Immunoprecipitation (IP) of large T antigen in SV40-transformed rat ovarian cells (DC3). DC3 cell lysates were immunoprecipitated with antibodies to p53 (G59-12, Cat. no. 14211A), SV40 large T antigen (PAb 100, Cat. no. 14141A) or with an IgG1 isotype control (clone A85-1, Cat. no. 02231D). Proteins were separated by SDS-PAGE and then western blotted (WB) with an antibody recognizing large T antigen (PAb 101, Cat. no. 14111A). The results show that SV40 large T could be immunoprecipitated with anti-large T antibody (PAb 100) or co-immunoprecipitated with an anti-p53 antibody (G59-12).

Antibodies to Rat Cell Biology Markers

GENES

Specificity

Clone

Application

Cat. No.

Format

Size

AKT AKT (P-Ser473) Androgen Receptor Androgen Receptor DBP E2F-1 FKBP-12 Jun Jun Ku-70/80 L1 Protein (HPV type 16) Latent TGFβ-1 BP MyoD PLC-γ1 PLC-γ2 PKC SC35 Sos1 Sp1 Stat1 TCP-1 TCP-1 TCP-1

polyclonal polyclonal G122-25

WB WB, IP FC, IHCF, IHCP

68601N 68591N 15051A

Se Se Pu

100 µl 100 µl 0.1 mg

G122-77

IP, IHCF

15071A

Pu

0.1 mg

10 KH95-E2F 2C1-87 G56-206 IKK-γ 162 CAMVIR

WB WB, IP WB, IHC WB WB IP, IF, FC, ELISA WB

14681A 14971A 13521A 13411A 68341A 66181A 14481A

Pu Pu Pu Pu Pu Pu Pu

0.1 0.1 0.1 0.1 0.1 0.1 0.1

polyclonal M0Ab5.8A polyclonal polyclonal MC5 alphaSC35 polyclonal 1C6 IF9A7 22B 84A 91A

IP, WB, IHCF, IHCP 15866E IP, WB, IHCF 13941A IP, WB 15526E IP, WB 15996E IP, WB, IHCF, IF 14871A IP, WB, IF 65201A WB, IHC 15191B WB 13931A WB, IP, IF 15221A IP, WB, IF 13321A IP, WB, IF 13311A IP, WB, IF 13331A

Se Pu Se Se Pu Pu Lyo Pu Pu Pu Pu Pu

100 µl 0.1 mg 100 µl 100 µl 0.1 mg 0.1 mg 0.2 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg

CELLS

mg mg mg mg mg mg mg

Antibodies to Rat Tumor Suppressors and Oncogene Markers Specificity

Clone

Application

Cat. No.

Format

Size

MOM2

SMP14

IHCF,

65101A

Pu

0.1 mg

p53

G59-12

IHCF,

14211A

Pu

0.1 mg

p53 p53 p53 FITC SET

PAb 122 PAb 240 G59-12, MOPC-21 G59-12, MOPC-21 DO-7,27-35 G3-245

IP, WB, IHCP WB, IP, IHCP WB, IP, WB, IP, FC

IF IHCF

14091A 14461A 6732KK

Pu Pu FITC

0.1 mg 0.1 mg 100 tests

FC

6733KK

PE

100 tests

FC WB, IP, FC, IF, IHCF, IHCP, GS FC

6680KK 14001A

PE Pu

100 tests 0.1 mg

6805KK

Cy-Chrome™

100 tests

FC

6684KK

FITC

100 tests

FC

6685KK

PE

100 tests

IP, IF, IHCF

14141A

Pu

0.1 mg

WB, IP, IF, IHCF

14111A

Pu

0.1 mg

p53 PE SET p53 PE SET Rb

Rb Cy-Chrome™ Set G3-245, MOPC-21 Rb FITC Set G3-245, MOPC-21 RB PE Set G3-245, MOPC-21 SV40 PAb-100 Large T Antigen SV40 PAb-101 Large T Antigen

Antibodies to Rat Cell Cycle Markers Specificity

Clone

Application

Cat. No.

Format

Size

CdK4 Cyclin A

DCS-35 BF683

Cyclin D1

G124-3216

Cyclin D1

DCS-6

WB,IP WB, IP WB, IP FC WB, IP WB, IP FC WB, IP

68941A 14531A 14531C 1370KK 14561A 14561C 1372KK 66271A

Pu Pu Pu FITC Set Pu Pu FITC Set Pu

0.1 mg 0.1 mg 0.25 mg 100 tests 0.1 mg 0.1 mg 100 tests 0.1 mg

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23

PROTEINS

GENES CELLS

PROTEINS

Antibodies to Rat Cell Cycle Markers, continued Specificity

Clone

Application

Cat. No.

Format

Size

Cyclin D3

G107-565

Cyclins D1/D2/D3

G124-259

WB, WB, FC WB, WB, FC

14781A 14781C 1374KK 14841A 14841C 1375KK

Pu Pu FITC Set Pu Pu FITC Set

0.1 mg 0.25 mg 100 tests 0.1 mg 0.25 mg 100 tests

IP IP IP IP

Antibodies to Rat Neurobiology Markers Specificity

Clone

Application

Cat. No.

Format

Size

14-3-3 Dopamine β-Hydroxylase GABA B Receptor GFAP (cocktail)

8C3 DBH-41

WB, IP WB

68291A 60101A

Pu Pu

0.1 mg 0.1 mg

polyclonal 4A11, 1B4,2E1 4A11 1B4 20 polyclonal

WB, IHCF WB, IF, IHCF, IHCP WB, IF, IHC WB, IF, IHC WB, IF, IHC WB, IP, IHCF

60696E 60341D

Se Pu

100 µl 0.5 mg

60311D 60321D 60331D 60656N

Pu Pu Pu Pu

0.5 mg 0.5 mg 0.5 mg 0.05 mg

polyclonal

WB, IP

60666N

Pu

0.05 mg

3A11

WB, IF, IHC

60011A

Pu

0.1 mg

54.1

WB, IHC

60021A

Pu

0.1 mg

Ap20 24B7.2D11 Myelin BP

WB, IHCF WB WB

60171A 68271A 69151A

Pu Pu Pu

0.1 mg 0.1 mg 0.1 mg

F5D

WB, GS, IF, IHCF, IHCP WB, GS WB, IHCF WB, IHCF WB, IHCF WB, IHCF

65121A

Pu

0.1 mg

65131A 60221A 60201A 60211A 60111A

Pu Pu Pu Pu Pu

0.1 0.1 0.1 0.1 0.1

WB, FC, IF, IHCF

60051A

Pu

0.1 mg

WB WB

60021A 69041G

Pu Se

0.1 mg .25 mg

WB

69021G

Se

.25 mg

WB

69031G

Se

.25 mg

WB

69051G

Se

.25 mg

WB, IHCF, IHCP

60151A

Pu

0.1 mg

WB, IHCP

60711A

Pu

0.1 mg

WB

60721A

Pu

0.1 mg

IP, IHC, EIA

60501A

Pu

0.1 mg

IHCF WB, IHCF

60121A 60231A

Pu Pu

0.1 mg 0.1 mg

WB, IHCF

60531A

Pu

0.1 mg

WB, IHCF

60541A

Pu

0.1 mg

GFAP GFAP GFAP Glutamate R (GluR2,R3) Glutamate R (GluR4) Glutamate R (GluR 5,6,7) Glutamate R (NMDAR1) MAP2 (a+b) MASH1 Myelin Basic Protein Myogenin

NF-YA YA-1a NCAM 12F8 Pan N-CAM 12F11 Pan N-CAM N-CAM 13 β Nerve NGF30 Growth Factor Nestin Rat 401 (Glutamate Receptor) NMDAR1 54.1 NMDAR1 (C2') polyclonal (Glutamate Receptor) NMDAR1 (C1) polyclonal (Glutamate Receptor) NMDAR1 (C2) polyclonal (Glutamate Receptor) NMDAR1 (N1) polyclonal (Glutamate Receptor) Tau Tau-2 (Tau-2 Epitope) (k) Opiod polyclonal Receptor (N-terminus) (k) Opiod polyclonal Receptor (internal region) (k) Opiod KA8 Receptor (N-terminus) Serotonin YC5/45 Serotonin G186-1117 Receptor (5-HT2AR) Serotonin A72-1 Receptor (5-HT2BR) Serotonin A4-2 Receptor (5-HT2CR)

24

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mg mg mg mg mg

Antibodies to Rat Neurobiology Markers, continued

GENES

Specificity

Clone

Application

Cat. No.

Format

Size

Somatostatin Substance P TrkA Tau (Tau-5 Epitope) β-Tubulin TRH,TYH, and PAH Hydroxylases Tyrosine Hydroxylase VAchT

YC7 NC1/34 polyclonal Tau-5

IHCF RIA, IHCF IHCF WB, IHCF, IHCP

60131A 60091A 68266E 60161A

Pu Pu Se Pu

0.1 mg 0.1 mg 100 µl 0.1 mg

5H1 PH8

WB, IF WB, IP, IHCF, IHCP

60181A 60061D

Pu Pu

0.1 mg 0.5 mg

TOH A1

IHCF

60081A

Pu

0.1 mg

polyclonal

IHCF

60596E

Se

100 µl

CELLS

Antibodies to Apoptosis Markers Specificity

Clone

Application

Bak Bax Bax Bcl-2 Bcl-2 Bcl-XL Bim E2F-1 Caspase-1 Caspase-2 Caspase-4 Cytochrome c Cytochrome c PARP PARP Raf Bak

polyclonal 6A7 polyclonal polyclonal polyclonal 2H12 polyclonal KH95/E2F B24-1 G310-1248 B25-1 6H2.B4 7H8.2C12 7D3-6 C-210 URP26K polyclonal

WB 65606E WB, IP 66241A WB, IP, IHCF, IHCP 13686E WB, IP, IHCP 13456E WB, IP, IHCP 15616E WB 66461A WB 68871N 14971A 14971A WB 66441A WB 13951A WB 66171A IP 65971A WB 65981A WB 66391A WB, IF 65196E IP, WB 13981A WB, IHCP 65606E

Cat. No.

Format

Size

Se Pu Se Se Se Pu Se Pu Pu Pu Pu Pu Pu Pu Asc Pu Pu

100 µl 0.1 mg 100 µl 100 µl 100 µl 0.1 mg 100 µl 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg 100 µl 0.1 mg 100 µl

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25

PROTEINS

GENES CELLS

PROTEINS

BD RiboQuant™ Ribonuclease Protection Assay (RPA) Systems The ribonuclease protection assay (RPA) is a highly sensitive and specific method to detect and quantitate RNA. The method is based on the hybridization of target RNA to 32P-labeled anti-sense RNA probes that have been in vitro-transcribed from a DNA template. RNase treatment follows, resulting in degradation of single-stranded RNA and excess probe. The probe and target RNA are resolved by denaturing polyacrylamide gel electrophoresis and the "protected" probe is visualized using autoradiography or phosphorimaging equipment. BD PharMingen’s Multi-Probe Template Sets are composed of a series of biologically related templates, each of distinct length and each representing a sequence in a distinct mRNA species.

Each template set is capable of detecting up to 11 unique gene transcripts in addition to two housekeeping genes, L32 and GAPDH. Samples of total RNA from various sources analyzed for distinct mRNA species using BD PharMingen’s RiboQuant™ Multi-Probe Ribonuclease Protection Assay System. The autoradiograms show the probe sets not treated with RNases (Lanes 1) along with the corresponding RNase-protected probes following hybridization with yeast tRNA (2 mg) (Lanes 2) and selected rat RNA (Lanes 3, 4, 5). Note that each undigested control probe band (Lanes 1) migrates more slowly than its protected and digested counterpoint (Lanes 3); this is due to the digestion of the flanking sequences in the probe that are not protected by the target mRNA. (Figures 84-88)

Rat RiboQuant™ Products Description

Size

Cat. No.

BD RiboQuant™ RPA Starter Package BD RiboQuant™ In Vitro Transcription Kit BD RiboQuant™ RNase Protection Assay Kit RNA Isolation Kit

10 rxns 25 rxns 200 rxns

45024K 45004K 45014K 45520K

10 rxns 10 rxns 10 rxns

45027P 45630P 45631P

Rat Cytokine Template Sets rCK-1 rCK-2 rCK-3

Rat Cytokine Template Set Rat Cytokine Template Set Rat Cytokine Template Set

Rat Neurobiology Template Sets rNT-1 Rat Neurotrophin Template Set

45028P

Rat Apoptosis Template Sets rAPO-1 RiboQuant™ Rat Apoptosis Template Set

26

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45601P

rAPO-1 (Cat. No. 45601P) Template

Probe

FAS bcl-X (L) bcl-X (S) FASL caspase-1 caspase-3 caspase-2 bax bcl-2 caspase-2 L32 GAPDH

FasL caspase-1 caspase-3 caspase-2 bax bcl-2

L32

406 363 (L) 325 (S) 286 253 226 202 (L) 181 160 137 (S) 112 97

2

3

4

GDNF CNTF NT3 NT4

rNT-1 (Cat. No. 45028P) Template

Probe

Protected

βNGF BDNF GDNF CNTF NT3 NT4 L32 GAPDH

351 315 282 255 231 213 141 126

322 286 253 226 202 184 112 97

GENES CELLS

PROTEINS

L32

GAPDH

Rat Control RNA

Probe

435 393 393 315 282 255 231 210 189 231 141 126

Protected

βNGF BDNF

Probe

GAPDH

Yeast tRNA

rNT-1 1

3

Figure 84

Rat Brain RNA (5 µg)

2

Rat Brain RNA (15 µg)

1

Yeast tRNA

rAPO-1 Fas bcl-x L/S

Figure 85

rCK-3 (Cat. No. 45631P)

rCK-3 1

2

3

1

Template

IFNβ TNFβ GM-CSF TGFβ1 TGFβ3 TGFβ2 Ltβ TNFα MIF IFNγ L32 GAPDH

TGFβ1 TGFβ3 TGFβ2 Ltβ TNFα MIF IFNγ L32

390 351 324 285 255 231 210 189 171 158 141 126

361 322 295 256 226 202 181 160 142 127 112 97

Template

IL-1α IL-1β IL-1Ra IL-12p40 IGIF/IL-18 IL-6 IL-10 MIF IFNγ L32 GAPDH

IL-18 IL-6 IL-10

MIF IFNγ L32 GAPDH

Probe

Protected

432 390 351 285 255 231 210 171 158 141 126

403 361 322 256 226 202 181 142 127 112 97

Probe Yeast tRNA Rat Bone Marrow Rat Control RNA

Figure 87

rCK-1 2

3

4

5

IL-1α IL-1β TNFβ IL-3 IL-4 IL-5 IL-6 IL-10 TNFα IL-2 IFNγ L32

CD8

αCD3: + αCD28: -

CD4

CD4

Yeast tRNA

GAPDH

Probe

3

IL-12p40

Figure 86

1

2

IL-1α IL-1β IL-1Ra

Probe Yeast tRNA Rat Control RNA

GAPDH

Probe Protected

rCK-2 (Cat. No. 45630P)

rCK-2

4

IFNβ TNFβ GM-CSF

+ +

+ +

rCK-1 (Cat. No. 45027P) Template

Probe

Protected

IL-1α IL-1β TNFβ IL-3 IL-4 IL-5 IL-6 IL-10 TNFa IL-2 IFNg L32 GAPDH

432 390 351 315 285 255 231 210 189 171 156 141 126

403 361 322 286 256 226 202 181 160 142 127 112 97

Special thanks to Dr. Frans Kroese for his technical contribution to the development of this brochure.

T-Cell

Figure 88

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The 2000 BD Biosciences Life Science Research Catalogs feature products from:

Who is BD Biosciences? BD Biosciences is made up of the following brands you know and trust:

• Becton Dickinson Immunocytometry Systems

PharMingen

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BD Immunocytometry Systems

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Transduction Laboratories

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Falcon™ BioCoat™

Im

B

m

un

ocyt

ometr

y S ys

te

D

m

BD Microbiology Systems

s

Biometric Imaging and most recently Clontech

20 0 0 B

D

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These are the things you associate with the name PharMingen and the products and services PharMingen provides. PharMingen has a new “official” name, but behind it is the same technical support and cutting edge scientific leadership you’ve trusted for more than a decade. What has changed is this – PharMingen is now proud to be part of the quality worldwide team by the name of BD Biosciences.

Products Catalog

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PharMinge

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In our business, a lot relies on a name. A name inspires confidence, it is the badge of the business you trust to provide consistently high quality products; lot after lot and year after year.

N

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BD PharMingen will continue to provide cutting edge reagents and quality technical service worldwide, while better serving your needs with a more complete BD Biosciences product portfolio. Your patience is greatly appreciated during this positive transition.

S

Research Products Catalog

Tr

B

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sd

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at

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B

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Research Products Catalog Includes BD PharMingen Cell Biology Products

I

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The catalog volumes are complete with a color-coded index, internal product cross-referencing, and the same detailed products descriptions and protocols you depend on.

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