rat immunology - from genes to proteins to cells · pdf file 1 introduction the rat is the...

Rat ImmunologyFrom Genes to Proteins to Cells

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Table of ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

CellsB 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

ProteinsCytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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

GenesBD RiboQuant™ Ribonuclease Protection Assay (RPA) Systems . . . . . . . 26

GENES

CELLS PROTEINS

www.bdbiosciences.com1

IntroductionThe rat is the second most frequentlyused animal, after the mouse, in thefield of immunological research.Traditionally, rats have been usedas experimental animals to studybehavior, physiology, and pathology,including tumor biology, transplan-tation, and toxicology. Togetherwith mice and guinea pigs, ratsbelong to the largest order ofmammals, the Rodentia. The lab-oratory rat was developed from thebrown rat, Rattus norvegicus, at thebeginning of the twentieth century,and most laboratory rat strainsoriginate from animals held at theWistar Institute of Anatomy andBiology in Philadelphia, USA.

A large number of genetically well-defined inbred laboratory rat strainsare now available for experimentalresearch. In addition, a variety ofcongenic, mutant and recombinantrat strains of immunological interesthave been developed in recent years,further expanding the possibilitiesof experimentation. These strainsinclude nude (athymic) rats, ratsthat spontaneously develop diabetes,and pairs of rats congenic for MHCclass I (RT1A) or II (RT1B andRT1D) haplotypes, immunoglobulinkappa light chain (Igκ-1) allotypesor the leukocyte common allo-antigen (RT7). BD PharMingenoffers antibodies that recognizepolymorphic and non-polymorphicepitopes of those alloantigensystems. (Please see the Rat MHCHaplotype chart on page 11.) Although rats are small animals,they are big enough to allow a widevariety of experimental manipula-tions. Their comparably larger size,than mice, offers unique possibilitiesfor the application of microsurgicaltechniques, therefore rats are often

the species of premier choice fortransplantation studies. For example,the recent technology to induceindefinite allograft survival byintrathymic injection of alloantigenwas first developed in rats. Rats arealso used frequently for the genera-tion of hybridomas and monoclonalantibodies. There are many ratautoimmune disease models whichdevelop spontaneously after immu-nization with appropriate antigens,after chemical treatments, or uponintroduction of a transgene. Someexamples of frequently used ratmodels of human autoimmunediseases are:

• Insulin-dependent diabetesmellitus (IDDM). Diabetes-pronebio-breeding (DP-BB) rats sponta-neously develop autoimmuneinsulin-dependent diabetes andthyroiditis at very high frequency.Hyperglycemia develops when ratsare between 50 and 90 days old andis associated with infiltration of theislets of Langerhans and selectivedestruction of pancreatic β cells.

• Multiple sclerosis. A widely usedanimal model for multiple sclerosis isexperimental autoimmune encepha-lomyelitis (EAE). EAE can beinduced relatively easily in Lewis ratsby inoculation with homogenizedcentral nervous system myelin orpurified myelin antigens togetherwith incomplete Freund's adjuvant.Both acute and chronic relapsingEAE models have been developed.

• Rheumatoid arthritis.Immunization of rats with myco-bacteria suspended in oil results inthe development of adjuvant arthritis,an extensively used animal model ofrheumatoid arthritis in humans.

• Glomerulonephritis. Injection ofBN rats with multiple low doses ofHgCl2 leads to the development ofchemically-induced autoimmuneglomerulonephritis associated withproteinurea and depositions ofautoantibodies to the renal glomerularbasement membrane.

Many monoclonal antibodies to ratcell surface antigens and solublefactors have been developed in thelast twenty years, and their numberis rapidly growing. The availabilityof these antibodies greatly facilitatesstudies of the immune system in therat. Many homologues of humanCluster of Differentiation (CD) antigens have been cloned in the ratallowing detailed analysis of relevant molecules of immunologicalimportance.

Undoubtedly, experimentation inthe rat will continue to be a criticaltool in biomedical research. Here,we have summarized selectedreagents and techniques availablefrom BD Pharmingen for immuno-logical research in the rat animalmodel.


B and T cells comprise the twomajor populations of small lymphocytes. B cells mature in thebone marrow and are defined bythe expression of the B cell receptorcomplex (BCR). The BCR consistsof membrane bound antibody molecules (sIg or mIg) complexedwith the Igα (CD79a) and Igβ(CD79b) molecules. While sIg recognizes and binds antigen,CD79a and CD79b participate inBCR signal transduction.

B cells generate the humoralimmune response and can functionas antigen presenting cells for T cell

activation and thus also play a rolein cell mediated immune response.One major difference between ratB cells and those in the human ormouse is that in the rat a CD5+

subset of B cells has not yet beendescribed. Mature B cells in the ratcan easily be distinguished fromother leukocytes either on the basisof their expression of sIg or on thebasis of their expression of CD45isoforms unique to B cells. TheseB cell isoforms of CD45 includethe CD45R isoform recognizedby HIS24 mAb and CD45RA recognized by OX-33 mAb.

B Lymphocytes

Figure 3 Figure 4

Figure 6

CD45RA-FITC (OX-33)

CD

80-P

E (

3H5)

B

CD45RA-FITC (OX-33)

CD

80-P

E (

3H5)

A

Figure 1,2. Activation-induced changesin the level of expression of the CD80antigen on fresh splenocytes (A) andcells treated in vitro for 72 hours withlipopolysaccharide (LPS) (B). Cells werestained simultaneously with CD45RA-FITC (OX-33) and CD80-PE (3H5).

GENES

CELLS PROTEINS

Isotype Control-FITC (G155-178)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

RT1A-FITC (OX-18)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

RT1B-FITC (OX-6)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

Ig light chain κ-FITC (MRK-1)

100

101

102

103

104

Figure 5

Figure 1

Figure 2


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

Figure 8

GENES

CELLS PROTEINS

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

IgM-FITC (G53-239)

100

101

102

103

104

CD5-FITC (OX-19)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

CD32-FITC (D34-485)

100

101

102

103

104

Marginal Zone B Cell Marker-FITC (HIS57)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

CD32 (D34-485) on paraffin-embedded spleen

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

Figure 7

Figure 9

Figure 11

Figure 10

Figure 12


Ms IgG1, κ-FITC (A112-2)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

NKR-P1A-FITC (10/78)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

Ms IgG1, κ-FITC (A112-2)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

NKR-P1A-FITC (10/78)

100 101 102 103 104

CD

45R

A-P

E (O

X-3

3)

100

101

102

103

104

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

Figures 13-16. Blocking of aggregated Ms IgG1, κ-FITC (A112-2) and anti-NKR-P1A-FITC (10/78) on ratsplenocytes by Rat FcBlock™ (CD32, D34-485). A112-2 and 10/78 lots selected for testing contained2-4% aggregates and approximately 4% of degraded products. FcR-mediated binding of aggregatesto 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™) (Figures13,14).

NEW Rat FcBlock™B cells also express Fcγ receptors(FcγR) belonging to theimmunoglobulin superfamily. Themajor Fcγ receptors in humans,mice, and rats are FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16).These receptors bind to the Fcregion of the IgG to help clearimmune complexes.

While CD64 binds monomeric IgG,CD32 and CD16 bind to aggregatedIgG.

In addition to Mouse FcBlock™,anti-mouse CD32/16, BD PharMingennow offers Rat FcBlock™ (anti-ratCD32) capable of blocking FcγR-mediated binding of aggregated IgGto FcγR bearing cells.

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

GENES

CELLS PROTEINS

Figure 13

Figure 15 Figure 16

Figure 14


GENES

CELLS PROTEINST cells, so named because theymature in the thymus, are a class ofsmall lymphocytes that can be distinguished from otherhematopoietic cells based on theirfunction and expression of the Tcell receptor (TCR). The TCR complex consists of a dimerichypervariable T cell receptor, whichbinds antigen, and an associatedinvariant CD3 complex, which isinvolved in TCR signal transduction. CD3-specific monoclonal antibodies (mAb) arewell suited as markers for mature Tcells and in combination with multi-color immunofluorescence stainingand flow cytometric analysis, are

used to study the expression patternof leukocyte antigens on T cells.Subsets of mature T cells can bedelineated by the expression ofeither CD4 or CD8, and these subsets can be further subdividedaccording to the types of cytokinesthe T cells secrete. T cells areimportant mediators of cytotoxicityand help to coordinate cell mediatedand humoral immune responses.

T cells can be classified not onlybased on the expression of CD4 orCD8 and cytokine secretion profilesbut also on the specific alpha orbeta chain of TCR they bear ontheir surfaces.

T Lymphocytes

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

Cel

l Nu

mb

er

CD25-FITC (0X-39)

Cel

l Nu

mb

er

CD134-FITC (OX-40)

Figures 21, 22. Expression of CD25 (IL-2Ralpha chain) and CD134 (OX-40) on ratspleen T cells (CD3+) following in vitroincubation for 48 hours with mediumalone (red line) or activation for 48hours with Concanavalin A (green line).

TCR αβ-FITC (R73)

CD

3-A

PC

(1F

4)

Figure 17

TCR γδ-FITC (V65)

CD

3-A

PC

(1F

4)

Figure 18

TCR Vα 8-FITC (G177)

CD

3-A

PC

(1F

4)

Figure 19

TCR Vβ 8.2/8.4-FITC (R78)

CD

3-A

PC

(1F

4)

Figure 20


Figures 23-35. Flow cytomet-ric data of rat peripheralblood cells stained with theCD3-APC (1F4) to identify Tcells and a panel of FITC-con-jugated antibodies (or bio-tinylated mAb + SAV-FITC)that react with cell-surfaceproteins of interest on T cells.The same antibody (in puri-fied format) was used forimmunohistochemical stainingof rat tissue sections.

CD2 (OX-34) on frozen spleen

CD4 (OX-38) on frozenspleen

CD

3-A

PC

(1F

4)

CD2-FITC (OX-34)

CD

3-A

PC

(1F

4)

CD4-FITC (OX-35)

CD5 (OX-19) on frozenspleen

CD5-FITC (OX-19)

CD

3-A

PC

(1F

4)

Figure 23

Figure 24

Figure 25

Figure 26 Figure 27

Figure 34 Figure 35

Figure 28 Figure 29

Figure 30 Figure 31


CD

3-A

PC

(1F

4)

CD8a-FITC (OX-8)

CD

3-A

PC

(1F

4)

CD8a (OX-8) on paraffin-embedded spleen

CD43 (HIS17) on paraffin-embedded spleen

CD43-FITC (HIS17)

CD

3-A

PC

(1F

4)

GENES

CELLS PROTEINS

Figure 32 Figure 33

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

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

CD

3-A

PC

(1F

4)


NK cells are large granular lympho-cytes that do not express either aBCR or a TCR and are capable ofnon-MHC restricted cell mediatedcytotoxicity without prior sensitiza-tion or coating of the target cellwith antibody. NK cells are also theeffector cells for antibody dependentcell mediated cytotoxicity, a process

in which cells coated with antibodyare destroyed following NK celldegranulation and release of perforinand granzymes. NK cells in the ratcan be distinguished either by highlevels of NKR-P1A expression or bythe fact that the majority of NKcells express CD8a, but do notco-express CD3.

NK Cells

Figure 36 Figure 37

Figure 38 Figure 39

Figure 40

Figures 36-40. Rat peripheral bloodcells stained with 10/78, a mAbspecific for NKR-P1A, and a panel ofFITC-conjugated antibodies thatreact with cell-surface proteins ofinterest on NK cells.

CD43-FITC (HIS17)

NK

R-P

1A-P

E (

10/7

8)

CD54-FITC (1A29)

NK

R-P

1A-P

E (

10/7

8)


NK

R-P

1A-P

E (

10/7

8)

CD2-FITC (OX-34)

NK

R-P

1A-P

E (

10/7

8)

CD3-FITC (1F4)

NK

R-P

1A-P

E (

10/7

8)

GENES

CELLS PROTEINS

NKR-P1A (10/78), frozen spleen

Figure 41. IHC staining of rat spleensection using the 10/78 mAb.


Monocytes/MacrophagesMonocytes and macrophagesconstitute a second major class ofleukocytes. Macrophages play animportant role in immune responsedue to their phagocytic properties,their ability to secrete cytokines andchemokines, and their ability tofunction as antigen presenting cells.Monocytes differ from macrophagesin that monocytes are immaturecirculating precursors to the varioustypes of tissue associated macrophages.Rat blood monocytes can bedistinguished from other leukocytesusing an antibody such as HIS48(specific for monocyte and granulocytelineages) combined with a gatingscheme that takes advantage of the

differential light scatteringproperties of granulocytes andmonocytes/lymphocytes (see nextsection). A second means foridentifying rat monocytes is basedon the fact that these cells expressCD4 but do not coexpress CD3.This scheme allows effectiveanalysis of the monocytic lineageutilizing BD PharMingen’s broadrange of antibodies for rat leukocytes.

Following the migration ofmonocytes to a tissue and theirsubsequent maturation, phenotypicchanges are observed on maturemacrophages (Figures 42,43).

Figures 44-51. Flow cyto-metric staining of severalcell surface antibodies torat peripheral bloodmonocytes characterizedby the expression of CD4(OX-35) and lack of CD3(1F4).


100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

CD11a-FITC (WT.1)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

CD11b-FITC (WT.5)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

100 101 102 103 104

CD

3-A

PC (

1F4)

CD4-PE (OX-35)

100

101

102

103

104

CD4- T cellsCD4+ T cells

CD3- CD4+Monocytes

Figure 44

Figure 47

Figure 50 Figure 51

Figure 48 Figure 49

Figure 45 Figure 46

CD61-FITC (F11)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

Granulocyte Marker-FITC (HIS48)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

CD80-FITC (3H5)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

CD86-FITC (24F)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

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

RT1B-FITC (OX-6)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

Blood CD3- CD4+ cells

RT1B-FITC (OX-6)

100 101 102 103 104

Cel

l Nu

mb

er

010

2040

5030

Spleen CD3- CD4+ cells

GENES

CELLS PROTEINS

Gated on Monocyte Population


GranulocytesGranulocytes comprise the lastmajor class of hematopoietic cells tobe discussed here. Granulocytescharacteristically possess numerouscytoplasmic granules. These cellsplay a role in inflammatory responses

and are capable of phagocytizingopsonized debris. Granulocytes canbest be distinguished from otherleukocytes when using flow cyto-metric analysis by their uniquelyhigh levels of side light scatter.

Figure 52

Figure 54

Figure 56 Figure 57

Figure 55

Figure 53

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

Forward Scatter

0 50 100 150 250

Sid

e Sc

atte

r

050

100

150

250

200

200

Granulocytes

Lymphocytes/ Monocytes

Isotype Control-APC (G155-178)

100 101 102 103 104

Cel

l Nu

mb

er

030

6012

015

090

CD11a-APC (WT.1)

100 101 102 103 104

Cel

l Nu

mb

er

060

120

240

300

180

CD11b-APC (WT.5)

100 101 102 103 104

Cel

l Nu

mb

er

060

120

240

300

180

CD61-APC (F11)

100 101 102 103 104

Cel

l Nu

mb

er

060

120

240

300

180

Granulocyte Marker-APC (HIS48)

100 101 102 103 104

Cel

l Nu

mb

er

060

120

240

300

180

GENES

CELLS PROTEINS

Gated on Granulocyte Population


Cell-Surface Adhesion MoleculesAdhesion molecules play an important role in several aspects ofan immune response. These molecules control the homing andmigration of leukocytes and mediatemany of the direct intercellularinteractions essential for successfulimmune responses. Families ofadhesion molecules involved inimmune responses include the integrins, the selectins, and membersof the Ig superfamily.

IntegrinsThe integrins are a large and diversefamily of cell surface molecules thatmediate interactions of cells withcomponents of the extracellularmatrix, as well as interactionsbetween cells. Integrins are noncovalently linked heterodimersconsisting of a large α chain (forexample CD11a, b, c and CD49a,b, d, c) paired with a smaller βchain (for example CD18, CD29,and CD61). Much of the diversitywithin the integrin family is generatedby the association of different α andβ integrins to form unique integrinheterodimers. These molecules playan important role in inflammatoryand immune responses (Figures 58-63).

SelectinsThe selectins are a family of carbo-hydrate binding cell adhesion mole-cules that play an important role inleukocyte homing. L-selectin(CD62L) is expressed on nearly allcirculating leukocytes and isinvolved in leukocyte homing viainteractions with vascularaddressins. P-selectin (CD62P) isexpressed on activated platelets andendothelial cells where it mediatesthe adhesion of neutrophils andmonocytes to the activated cells.E-selectin (CD62E) is expressed onactivated endothelium where it alsoplays a role in the migration ofimmune cells (Figure 64).

Ig SuperfamilyA third family of adhesion mole-cules are those which are membersof the immunoglobulin (Ig) super-family. The Ig superfamily includesover 100 members many of whichare adhesion molecules (for exampleCD2, CD31, CD54, and CD106)(Figures 65-66 on page 11).

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

Figure 58 Figure 59

Figure 60 Figure 61

Figure 62 Figure 63

CD18-FITC (WT.3)

Cel

l Nu

mb

er

CD18 (WT.3) on paraffin-embedded spleen

CD29 (Ha2/5) on frozen spleen

CD29-FITC (Ha2/5)

CD

3-P

E (

1F4)

CD29 (Ha2/5) on frozen spleen

CD62L-FITC (HRL1)

CD

3-P

E (

1F4)

GENES

CELLS PROTEINS

CD11a-FITC (WT.1)

Cel

l Nu

mb

er

Figures 58-63. Flow cytometric and IHC data generatedfrom rat spleen using antibodies specific for theindicated members of the integrin family.

CD11a (WT.1) on frozen spleen


GENES

CELLS PROTEINS

Rat MHC Haplotype ChartRT1 Haplotype Strainsa AVNav1 DA, ACI, ACP, COPb BUF, ALBc AUG, PVGd BVDdv1 BDIX, TALe BDVIIf AS2g KGHh HWi BIj LEJk WKA/Hok, WKAH, KYN, OKA, SHRl LEWlvl F344m MNRn BN, MAXXo MRp RPq NIG IIIsa NSDsb WiNu WF, TO, AO, LOU, WAG, SDJ, YO, BB/W, DZB

Antibodies to Rat Leukocytes and Related Cells

Specificity Clone Isotype Format Size Cat. No.CD2 (LFA-2) OX- 34 Mouse IgG2a Purified 0.5 mg 22001D

FITC 0.5 mg 22004DPE 0.1 mg 22005A

CD3 G4.18 Mouse IgG3 NA/LE™ 0.5 mg 22010DPurified 0.5 mg 22011DBiotin 0.5 mg 22012DFITC 0.5 mg 22014DPE 0.2 mg 22015B

CD3 1F4 Mouse IgM Purified 0.5 mg 22731DFITC 0.5 mg 22734DPE 0.1 mg 22735AAPC 0.1 mg 22739A

CD4 OX-35 Mouse IgG2a Purified 0.5 mg 22021DBiotin 0.5 mg 22022DFITC 0.5 mg 22024DPE 0.2 mg 22025BCy-Chrome™ 0.1 mg 22028AAPC 0.1 mg 22029A

CD4 OX- 38 Mouse IgG2a Purified 0.5 mg 22031DFITC 0.5 mg 22034DPE 0.2 mg 22035B

CD5 OX-19 Mouse IgG1 Purified 0.5 mg 22051DFITC 0.5 mg 22054DPE 0.2 mg 22055B

CD5 HIS47 Mouse IgG2a Purified 0.5 mg 22061D

CD8a OX-8 Mouse IgG1 Purified 0.5 mg 22071DBiotin 0.5 mg 22072DFITC 0.5 mg 22074DPE 0.2 mg 22075B

CD8a G28 Mouse IgG2a Purified 0.1 mg 22421AFITC 0.1 mg 22424A

CD8b 341 Mouse IgG1 Purified 0.5 mg 22501DBiotin 0.5 mg 22502DFITC 0.5 mg 22504D

Figures 65, 66. Flow cytometric andIHC data generated from rat spleenusing antibodies specific for adhe-sion molecules belonging to the Igsuperfamily.

Figure 66

MAdCAM-1 (OST20) on paraffin-embedded spleen

Figure 65

CD54-FITC (1A29)

CD

3-A

PC

(1F

4)


Antibodies to Rat Leukocytes and Related Cells, continued

Specificity Clone Isotype Format Size Cat. No.CD9 RPM.7 Mouse IgG3 Purified 0.5 mg 22791D

FITC 0.5 mg 22794DCD11a WT.1 Mouse IgG2a Purified 0.1 mg 22541A(Integrin αL chain FITC 0.5 mg 22544DLFA-1α chain) PE 0.2 mg 22545BCD11b WT.5 Mouse IgA Purified 0.5 mg 22531D(Integrin aM chain, Biotin 0.5 mg 22532DMac-1 α chain) FITC 0.5 mg 22534DCD11b/c OX-42 Mouse IgG2a NA/LE™ 0.5 mg 22080D

Purified 0.5 mg 22081DBiotin 0.5 mg 22082DFITC 0.5 mg 22084DPE 0.2 mg 22085B

CD15s 2H5 Mouse IgM Purified 0.1 mg 33601A[Sialyl Lewis X (SLex)]CD18 WT.3 Mouse IgG1 NA/LE™ 0.5 mg 22520D(Integrin β2 chain) Purified 0.5 mg 22521D

FITC 0.5 mg 22524DCD25 OX - 39 Mouse IgG1 Purified 0.1 mg 22091A(IL-2R α chain) Biotin 0.1 mg 22092A

FITC 0.5 mg 22094DPE 0.2 mg 22095B

New CD26 OX-61 Mouse IgG2a Purified 0.1 mg 22811AFITC 0.1 mg 22814APE 0.1 mg 22815A

New CD27 LG.3A10 Hamster IgG1 NA/LE™ 0.5 mg 09630DPurified 0.1 mg 09631ABiotin 0.1 mg 09632APE 0.1 mg 09635A

CD28 JJ319 Mouse IgG1 NA/LE™ 0.5 mg 22600DPurified 0.1 mg 22601ABiotin 0.1 mg 22602AFITC 0.5 mg 22604DPE 0.1 mg 22605A

CD28 JJ316 Mouse IgG1 NA/LE™ 0.5 mg 22590D

CD29 Ha2/5 Armenian NA/LE™ 0.5 mg 22630D(Integrin β1 chain) Hamster IgM Purified 0.5 mg 22631D

Biotin 0.5 mg 22632DFITC 0.5 mg 22634D

CD29 HMβ1-1 Armenian Purified 0.5 mg 09871D(Integrin β1 chain) Hamster

IgG, Group 2CD31 (PECAM-1) TLD-3A12 Mouse IgG1 NA/LE™ 0.5 mg 22710D

Purified 0.5 mg 22711DBiotin 0.5 mg 22712DPE 0.1 mg 22715A

New CD32 D34-485 Mouse IgG1 NA/LE™ 0.5 mg 22880DPurified 0.5 mg 22881DPurified 0.1 mg 22881AFITC 0.5 mg 22884D

New OX-40 ATM-2 Mouse IgG1 Purified 0.5 mg 22821DLigand Biotin 0.5 mg 22822DCD42d RPM.4 Mouse IgG2a Purified 0.5 mg 22771D

FITC 0.5 mg 22774DCD43 HIS17 Mouse IgG1 Purified 0.5 mg 22101D(Leukosialin) Biotin 0.5 mg 22102DCD44 H OX-49 Mouse IgG2a Purified 0.5 mg 22111D(Pgp-1, H-CAM) FITC 0.5 mg 22114D

PE 0.2 mg 22115BCD45.2 HIS41 Mouse IgG1 Purified 0.1 mg 22121A(RT7.2 of Leukocyte Common Antigen) FITC 0.1 mg 22124ACD45 OX-1 Mouse IgG1 Purified 0.5 mg 22131D(Leukocyte Common Antigen) Biotin 0.5 mg 22132D

FITC 0.5 mg 22134DPE 0.2 mg 22135BCy-Chrome™ 0.1 mg 22138A

GENES

CELLS PROTEINS



Specificity Clone Isotype Format Size Cat. No.CD45R HIS24 Mouse IgG2b Purified 0.5 mg 22161D

FITC 0.5 mg 22164DPE 0.2 mg 22165B

CD45RA OX-33 Mouse IgG1 Purified 0.5 mg 22171DBiotin 0.5 mg 22172DFITC 0.5 mg 22174DPE 0.2 mg 22175BCy-Chrome™ 0.1 mg 22178A

CD45RC OX-22 Mouse IgG1 Purified 0.5 mg 22181DFITC 0.5 mg 22184DPE 0.2 mg 22185B

CD45RC HIS25 Mouse IgG1 Purified 0.5 mg 22221D

CD49a Ha31/8 Armenian NA/LE™ 0.5 mg 22620D(Integrin α1 chain) Hamster Purified 0.5 mg 22621D

IgG, Group 2CD49b Ha1/29 Armenian Purified 0.5 mg 22611D(Integrin α2 chain) Hamster FITC 0.5 mg 22614D

IgG, Group 2CD49d MRα4-1 Mouse IgG2a Purified 0.5 mg 01871D(Integrin α4 chain) FITC 0.1 mg 01874ACD49e HMα5-1 Armenian Purified 0.5 mg 01881D(Integrin α5 chain) Hamster

IgG, Group 1CD53 OX-44 Mouse IgG1 Purified 0.5 mg 22751D

FITC 0.5 mg 22754DCD54 (ICAM-1) 1A29 Mouse IgG1 NA/LE™ 0.5 mg 22490D

Purified 0.5 mg 22491DBiotin 0.5 mg 22492DFITC 0.5 mg 22494DPE 0.2 mg 22495B

CD59 TH9 Mouse IgG1 Purified 0.5 mg 22721DFITC 0.5 mg 22724D

CD61 F11 Mouse IgG1 NA/LE™ 0.5 mg 22440D(Integrin β3 chain) Purified 0.5 mg 22441D

FITC 0.5 mg 22444DCD61 2C9.G2 Armenian NA/LE™ 0.5 mg 01860D(Integrin β3 chain) Hamster Purified 0.5 mg 01861D

IgG, Group 1 Biotin 0.5 mg 01862DFITC 0.5 mg 01864DPE 0.2 mg 01865B

CD62L HRL1 Armenian Purified 0.1 mg 22471A(L-selectin, LECAM-1) Hamster Biotin 0.1 mg 22472A

IgG, Group 2 FITC 0.5 mg 22474DPE 0.2 mg 22475B

CD62L HRL2 Armenian Purified 0.1 mg 22481A(L-selectin, LECAM-1) Hamster

IgG, Group 1CD62P Polyclonal Rabbit IgG Purified 0.1 mg 09361A(P-selectin)CD71 OX-26 Mouse IgG2a Purified 0.5 mg 22191D(Transferrin Receptor) FITC 0.5 mg 22194D

PE 0.2 mg 22195BCD80 (B7-1) 3H5 Mouse IgG1 NA/LE™ 0.5 mg 22660D

Purified 0.5 mg 22661DBiotin 0.5 mg 22662DPE 0.2 mg 22665B

CD86 (B7-2) 24F Mouse IgG1 Purified 0.5 mg 22671D

CD90 (Thy-1) HIS51 Mouse IgG2a Purified 0.5 mg 22201DBiotin 0.5 mg 22202DFITC 0.5 mg 22204D

CD90 (Thy-1) OX-7 Mouse IgG1 Purified 0.5 mg 22211DBiotin 0.5 mg 22212DFITC 0.5 mg 22214DPE 0.2 mg 22215BPerCP 0.1 mg 2221PA

GENES

CELLS PROTEINS



Specificity Clone Isotype Format Size Cat. No.CD122 L316 Mouse IgG1 Purified 0.5 mg 22741D(IL-2Rβ chain) Biotin 0.5 mg 22742D

FITC 0.5 mg 22744DCD134 OX-40 Mouse IgG2b Purified 0.1 mg 22041A(OX-40 Antigen) Biotin 0.5 mg 22042D

FITC 0.5 mg 22044Dβ2 Microglobulin TLD-3H12b Mouse IgG1 Purified 0.5 mg 22801D

FITC 0.5 mg 22804DCrry/p65 512 Mouse IgG1 Purified 0.5 mg 22581D

Dendritic Cells OX-62 Mouse IgG1 Purified 0.5 mg 22651DFas Ligand MFL4 Armenian NA/LE™ 0.5 mg 22700D

Hamster Purified 0.5 mg 22701DIgG, Group 3 Biotin 0.5 mg 22702D

Granulocytes HIS48 Mouse IgM Purified 0.5 mg 22261DBiotin 0.5 mg 22262DFITC 0.5 mg 22264D

New Granulocytes RP-1 Mouse IgG2a Purified 0.5 mg 22851DBiotin 0.5 mg 22852DPE 0.1 mg 22855A

New Granulocytes RP-3 Mouse IgM NA/LE™ 0.5 mg 22840DPurified 0.5 mg 22841D

Macrophage anti-RMA Mouse IgG1 Purified 0.5 mg 22691DActivatorMacrophage HIS36 Mouse IgG2a Purified 0.5 mg 22231DSubset PE 0.2 mg 22235BNew MAdCAM-1 OST2 Mouse IgG1 NA/LE™ 0.5 mg 22860D

Purified 0.1 mg 22861ANew Marginal HIS57 Mouse IgG1 Purified 0.1 mg 22831AZone B Cells Biotin 0.1 mg 22832A

FITC 0.5 mg 22834DMononuclear 1C7 Mouse IgG1 Purified 0.5 mg 22451DPhagocyte Biotin 0.1 mg 22452ANKR-P1A (CD161) 10/78 Mouse IgG1 Purified 0.5 mg 22641D

Biotin 0.5 mg 22642DFITC 0.5 mg 22644DPE 0.2 mg 22645B

Pan-T Cells OX-52 Mouse IgG2a Purified 0.5 mg 22251DFITC 0.5 mg 22254D

QCA-1 HIS45 Mouse IgG1 Purified 0.5 mg 22241D

New VCAM-1 MR106 Mouse IgG1 Purified 0.5 mg 22681D(CD106) PE 0.1 mg 22685A

Antibodies to Rat Major Histocompatability Complex (MHC) Antigens

Specificity Clone Isotype Format Size Cat. No.RT1A OX-18 Mouse IgG1 Purified 0.5 mg 22301D

Biotin 0.5 mg 22302DFITC 0.5 mg 22304DPE 0.1 mg 22305A

RT1A a, b C3 Rat IgG2b Purified 0.5 mg 22351DBiotin 0.1 mg 22352AFITC 0.5 mg 22354D

RT1B a, b, l B5 Rat IgM Purified 0.1 mg 22371AFITC 0.1 mg 22374A

RT1B OX-6 Mouse IgG1 NA/LE™ 0.5 mg 22380DPurified 0.5 mg 22331DBiotin 0.5 mg 22332DFITC 0.5 mg 22334DPE 0.2 mg 22335BPerCP 0.1 mg 2233PA

RT1B HIS19 Mouse IgG1 Purified 0.5 mg 22311D(non-n haplotypes)RT1D OX-17 Mouse IgG1 Purified 0.5 mg 22341D

FITC 0.5 mg 22344D

GENES

CELLS PROTEINS


Antibodies to Rat T-Cell Receptors (TCR)

Specificity Clone Isotype Format Size Cat. No.αβ TCR R73 Mouse IgG1 NA/LE™ 0.5 mg 22280D

Purified 0.5 mg 22281DBiotin 0.5 mg 22282DFITC 0.5 mg 22284DPE 0.2 mg 22285BPerCP 0.1 mg 2228PA

γδ TCR V65 Mouse IgG1 Purified 0.5 mg 22461DBiotin 0.1 mg 22462AFITC 0.5 mg 22464DPE 0.2 mg 22465B

γδ TCR Subset V45 Mouse IgG1 Purified 0.5 mg 22511DBiotin 0.5 mg 22512D

Vα 4 TCR G99 Mouse IgG1 Purified 0.5 mg 22561D

Vα 8 TCR G177 Mouse IgG1 Purified 0.5 mg 22551D

Vβ 8.21 R78 Mouse IgG1 Purified 0.5 mg 22391Dand Vβ 8.4a TCR FITC 0.1 mg 22394AVβ 8.5 TCR B73 Mouse IgG1 Purified 0.5 mg 22401D

Vβ 10 TCR G101 Mouse IgG2a FITC 0.5 mg 22414D

Vβ 16 TCR HIS42 Mouse IgG2b Purified 0.5 mg 22291D

Isotype Controls

Specificity Clone Format Size Cat. No.Mouse IgG1, κ MOPC-31C Purified 0.5 mg 03251D

Mouse IgG1, κ A112-2 NA/LE™ 0.5 mg 03210D(anti-KLH) Purified 0.5 mg 03211D

Biotin 0.25 mg 03212CFITC 0.25 mg 03214CPE 0.1 mg 03215ACy-Chrome™ 0.1 mg 03218APerCP 0.1 mg 0321PA

Mouse IgG1, κ A115-2 Purified 0.5 mg 03221D(b allotype, anti-KLH)Mouse IgG1, λ A111-3 Purified 0.5 mg 03191D(anti-TNP)Mouse IgG1, λ S1-68.1 Purified 0.5 mg 03011D

Mouse IgG2a, κ G155-178 NA/LE™ 0.5 mg 03020D(anti-TNP) Purified 0.5 mg 03021D

Biotin 0.25 mg 03022CFITC 0.25 mg 03024CPE 0.1 mg 03025ACy-Chrome™ 0.1 mg 03028A

Mouse IgG2a, λ HOPC-1 Purified 0.5 mg 03031D

Mouse IgG2b, κ 49.2 Biotin 0.25 mg 03042C(anti-TNP) FITC 0.25 mg 03044C

PE 0.1 mg 03045AMouse IgG2b, λ G11-59 Purified 0.5 mg 03051D

Mouse IgG3, κ J606 NA/LE™ 0.5 mg 03060D(anti-Fructosan)Mouse IgG3, κ A112-3 Purified 0.5 mg 03201D(anti-KLH) Biotin 0.25 mg 03202C

FITC 0.25 mg 03204CPE 0.1 mg 03205A

Mouse IgG3, λ G19-143 Purified 0.5 mg 03071D

Mouse IgM, κ G155-228 Purified 0.5 mg 03081D(anti-TNP) Biotin 0.25 mg 03082C

FITC 0.25 mg 03084CMouse IgM, λ MOPC-104E Purified 0.5 mg 03091D(anti-Dextran)Mouse IgA, κ M18-254 Purified 0.5 mg 03101D

Biotin 0.25 mg 03102CFITC 0.25 mg 03104C

GENES

CELLS PROTEINS


GENES

CELLS PROTEINS

Isotype Controls, continued

Specificity Clone Format Size Cat. No.Mouse IgA, λ MOPC-315 Purified 0.5 mg 03111DMouse IgE, κ 27-74 Purified 0.5 mg 03131D(anti-dansyl)Mouse IgE, κ C38-2 Purified 0.5 mg 03231D(anti-TNP)Mouse IgE, κ C48-2 Purified 0.5 mg 03241D(b allotype, anti-TNP)Hamster IgG, κ A19-3 NA/LE™ 0.5 mg 11150Dgroup 1, (anti-TNP) Purified 0.5 mg 11151D

Biotin 0.25 mg 11152CFITC 0.25 mg 11154CPE 0.1 mg 11155ACy-Chrome™ 0.1 mg 11158AAPC 0.1 mg 11159APerCP 0.1 mg 1115PA

Hamster IgG, λ G235-2356 NA/LE™ 0.5 mg 11120Dgroup 1, (anti-TNP) Purified 0.5 mg 11121D

Biotin 0.25 mg 11122CFITC 0.25 mg 11124CPE 0.1 mg 11125ACy-Chrome™ 0.1 mg 11128A

Hamster IgG, λ Ha4/8 NA/LE™ 0.5 mg 11140Dgroup 2, (anti-KLH) Purified 0.5 mg 11141D

Biotin 0.25 mg 11142CFITC 0.25 mg 11144CPE 0.1 mg 11145ACy-Chrome™ 0.1 mg 11148AAPC 0.1 mg 11149A

Hamster IgG, λ A19-4 NA/LE™ 0.5 mg 11160Dgroup 3, (anti-TNP) Purified 0.5 mg 11161D

Biotin 0.25 mg 11162CFITC 0.25 mg 11164CPE 0.1 mg 11165A

Hamster IgM, λ G235-1 NA/LE™ 0.5 mg 11130D(anti-TNP) Purified 0.5 mg 11131D

Biotin 0.25 mg 11132CFITC 0.25 mg 11134C


Detection of IntracellularCytokines by Flow Cytometry

GENES

CELLS PROTEINSThe development and function ofthe immune system depends on theproduction and response tocytokines. Cytokines are pleiotropicmediators capable of stimulatingmultiple biological effects.Cytokines have taken center stage inmany clinical and basic researchstudies because they play such a primary role in transmitting regulatory signals between varioustypes of cells. The intercellular communication system consisting ofcytokines and their receptors iscomprised of multiple stimulatoryand inhibitory cytokines. The complexity of the cytokine networkoften necessitates a multi-parameterapproach to study the functionalroles played by a particularcytokine, since each cytokine’s rolemay be influenced by othercytokines which are present in

the experimental system.BD PharMingen has designed itsline of cytokine reagents in responseto the needs of researchers forsimultaneous analysis of multiplecytokines.

BD PharMingen offers a growingline of reagents for the study of ratcytokines and chemokines includingrecombinant rat cytokines, specificantibodies to rat cytokines andchemokines, and optimizedimmunoassays. These antibodies areoffered in formats useful for detection of cytokine producingcells by flow cytometry, determination of cytokine proteinlevels by sandwich ELISA, as well as funcional analysis utilizing biologically active recombinant proteins, and neutralizing anti-cytokine antibodies.

Multicolor immunofluorescentstaining with antibodies againstintracellular cytokines and cell surface markers provides a high-resolution method to determine thenature and frequency of cells whichexpress a particular cytokine(s). Forexample, staining of an individualcell surface antigen and two cytoplasmic cytokines has been usedto identify and enumerate cell typesthat express cytokines in either apolarized (e.g., Th1- versus Th2-likecells) or unpolarized (e.g., Th0-likecells) pattern. In addition toenabling highly specific and sensitive measurements of severalparameters for individual cellssimultaneously, this method has thecapacity for rapid analysis of largenumbers of cells, which are

required for making statistically significant measurements.

In addition to fluorochrome-conjugated antibodies for intracellular staining of ratcytokines, BD PharMingen providesCytofix/Cytoperm Intracellular flowcytometry kits that providefixation and permeabilizationbuffers and protein transportinhibitors. Also now available areRiCK (Rat intracellular CytoKine)positive control cells. These cellsuspensions contain fixed, non-permeabilized rat lymphoid cellsthat express easily detected levels ofintracellular cytokines and can thusbe used as positive controls forexperiments.

CD4-FITC (OX-38)

Isot

ype

Con

trol-P

E (A

112-

2)

Figure 67

CD4-FITC (OX-38)

IL-4

-PE

(OX

-81)

11.8%

Figure 68

CD4-FITC (OX-38)

IL-1

0-P

E (A

5-4)

10.7%

Figure 69

CD4-FITC (OX-38)

GM

-CS

F-P

E (B

61-5

)

13.6%

Figure 70

Forward light scatter

Sid

e lig

ht s

catte

r

Figure 71

Cytokines

Figures 67-71. RiCK-2 positivecontrol cells stained with FITC-conjugated mouse anti-rat CD4(OX-38), washed, permeabi-lized, and subsequently stainedwith PE mouse IgG1 isotypecontrol (A112-2), PE-mouseanti-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 light-scattering characteristics forRiCK-2 cells are shown.


GENES

CELLS PROTEINS

Antibodies for Inracellular Cytokine and Chemokine Flow CytometryAntibodies for Rat Cytokines

Specificity Clone Isotype Format Application Size Cat. No.IL-4 OX-81 Mouse IgG1 Purified IC/Flow blocking 0.1 mg 24051A

PE IC/Flow staining 0.1 mg 24054AIFN-γ DB-1 Mouse IgG1 Purified IC/Flow blocking 50 ng 20441N

FITC IC/Flow staining 100 tests 20444XPE IC/Flow staining 100 tests 20445X

IL-10 A5-4 Mouse IgG2b Purified IC/Flow blocking 0.1 mg 24091APE IC/Flow staining 0.1 mg 24095A

TNF-α TN3-19.12 Hamster IgG Purified IC/Flow blocking 0.1 mg 23351APE IC/Flow staining 0.1 mg 23355A

GM-CSF B61-5 Mouse IgG1 Purified IC/Flow blocking 0.1 mg 24131APE IC/Flow staining 0.1 mg 24135A

Antibodies for Rat ChemokinesMCP-1 2H5 Hamster IgG Purified IC/Flow blocking 0.1 mg 18241A

PE IC/Flow staining 0.1 mg 18245A

Cell Fixation and Permeabilization Kits and ComponentsCytofix/Cytoperm™ Kit 250 tests 2075KKCytofix/Cytoperm™ Kit with GolgiStop™ 250 tests 2076KKCytofix/Cytoperm™ Kit with GolgiPlug™ 250 tests 2300KKGolgiStop™ (with Monensin) 1.0 ml 2092KZGolgiPlug™ (with Brefeldin A) 0.7 ml 2301KZCytofix/Cytoperm™ Buffer 100 mls 2090KZPerm/Wash™ Buffer 100 mls 2091KZNEW BrdU Flow Kit 50 tests 2354KK

Rat Intracellular Cytokine Positive Control CellsRiCK-2 (Positive for IL-4, IL-10, GM-CSF, IFN-γ, TNF-α) 5x10

6cells 24142Z

Antibodies for Rat Cytokine ReceptorsIL-2Rα OX-39 Mouse IgG1 Purified Flow Cytometry 0.5 mg 22091D

Biotin 0.5 mg 22092DFITC 0.5 mg 22094DPE 0.2 mg 22095B

IL-2Rβ L316 Mouse IgG1 Purified Flow Cytometry 0.5 mg 22741DBiotin 0.5 mg 22742DFITC 0.5 mg 22744D

Reagents for Immunofluoresent Staining of Cell SuspensionsPharMingenStain Buffer (with FBS) 20151EPharMingenStain Buffer (with BSA) 20161ECytofix™ Buffer 2014KZ

Neutralizing Rat Cytokine AntibodiesIL-2 Polyclonal Rabbit Ig NA/LE™ Neutralization 0.5 mg 24030DIL-4 OX-81 Mouse IgG1 NA/LE™ Neutralization 0.5 mg 24050D

TNF-α TN3-19.12 Hamster Ig NA/LE™ Neutralization 0.5 mg 23350C

Recombinant Rat Cytokines and ChemokinesIL-2 Bioactive/ELISA 5 µg 25001VIL-4 Bioactive/ELISA 5 µg 25011VIL-6 Bioactive/ELISA 5 µg 25081VIL-10 (ELISA Standard) ELISA only 5 µg 25071VGM-CSF Bioactive/ELISA 5 µg 25051VIFN-γ Bioactive 2 µg 25061VTNF-α Bioactive/ELISA 5 µg 25031VMCP-1 Bioactive/ELISA 5 µg 25041V


BD OptEIA™ Rat Cytokine ELISA Sets GENES

CELLS PROTEINSBD OptEIA™ ELISA Sets are specially formulated to facilitateaccurate measurement of solubleproteins in tissue culture super-natents, sera, plasma, and other complex biological fluids. Each setconsists of pre-titered capture anddetection antibodies along withlyophilized recombinant cytokinestandard and horseradish peroxi-dase conjugate sufficient for 20

96-well plates. The BD OptEIA™Set reagents are specifically formu-lated to reduce background andnon-specific binding caused by autoantibodies, rheumatoid factor,and plasma proteins such as complement, fibronectin and Fcreceptors. Typical standard curvesfrom BD OptEIA™ Sets for ratIL-2, IL-4, IL-10, IFN-γ, TNF-α,and MCP-1 are shown below.

Rat IL-10 OptEIA™ Set; Cat. No. 2611KIContents:• 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

0.01

0.1

1

Op

tica

l Den

sity

(45

0 n

m)

10 100 1000

IL-10 (pg/ml)

Rat IL -10 BD OptEIA™ Set

Rat IL-4 OptEIA™ Set; Cat. No. 2631KIContents:• Capture Antibody: Anti-rat IL-4• Detection Antibody: Biotinylated anti-rat• Enzyme regent: HRP-conjugated avidin• Standard: Recombinant rat IL-4


0.01

0.1

1

Opt

ical

Den

sity

(45

0 nm

)

1 10 100IL-4 (pg/ml)

Rat IL-4 BD OptEIA™ Set

Rat IL-2 OptEIA™ Set; Cat. No. 2628KIContents:• Capture Antibody: Anti-rat IL-2• Detection Antibody: Biotinylated anti-rat IL-2• Enzyme Reagent: HRP-conjugated avidin• Standard: Recombinant rat IL-2


0.01

0.1

1

Opt

ical

Den

sity

(45

0 nm

)

10 100 1000

IL-2 (pg/ml)

Rat IL-2 BD OptEIA™ Set

Rat IFN-γ OptEIA™ Set; Cat. No. 2696KIContents:• Capture Antibody: Anti-rat IFN-γ• Detection Antibody: Biotinylated anti-rat IFN-γ• Enzyme Reagent: HRP-conjugated avidin• Standard: Recombinant rat IFN-γ


Rat IFN-γ BD OptEIA™ Set

IFN-γ (pg/ml)

Op

tica

l Den

sity

(45

0 n

m)

Rat TNF-α OptEIA™ Set; Cat. No. 2697KIContents:• Capture Antibody: Anti-rat TNF-α• Detection Antibody: Biotinylated anti-rat TNF-α• Enzyme Reagent: HRP-conjugated avidin• Standard: Recombinant rat TNF-α


Rat TNF-α BD OptEIA™ Set

TNF-α (pg/ml)

Op

tica

l Den

sity

(45

0 n

m)

Rat MCP-1 OptEIA™ Set; Cat. No. 2610KIContents: • 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 0.01

0.1

1

Op

tica

l Den

sity

(45

0 n

m)

10 100 1000

MCP-1 (pg/ml)

Rat MCP-1 BD OptEIA™ Set

US Price 550


Matched Antibody Pairs and Protein Standards for Rat Cytokine and Chemokine ELISA

Specificity Description Clone Isotype Format Size Cat. No.IL-2 Capture Polyclonal Rabbit Ig Purified 0.5 mg 24031D

Detection A38-3 Mouse IgG1 Biotin 0.5 mg 24042DStandard Rec. IL-2 Recombinant 5 µg 25001V

IL-4 Capture OX-81 Mouse IgG1 Purified 0.5 mg 24051DDetection Polyclonal Rabbit Ig Biotin 0.5 mg 24112DStandard Rec. IL-4 Recombinant 0.5 µg 25011V

IL-4 Capture OX-81 Mouse IgG1 Purified 0.5 mg 24051DDetection B11-3 Mouse IgG1 Biotin 0.5 mg 24112DStandard Rec. IL-4 Recombinant 0.5 µg 25011V

IL-10 Capture A5-7 Mouse IgG1 Purified 0.5 mg 24061DDetection A5-5 Mouse IgG1 Biotin 0.5 mg 24072DStandard Rec. IL-10 Recombinant 0.5 µg 25071V

GM-CSF Capture B61-5/B61-9 Mouse IgG1 Purified 0.5 mg 24181DDetection B61-10 Mouse IgG1 Biotin 0.5 mg 24172DStandard Rec. GM-CSF Recombinant 0.5 µg 25051V

MCP-1 Capture C4 Mouse IgG1 Purified 0.5 mg 24011DDetection B4 Mouse IgG1 Biotin 0.5 mg 24022DStandard Rec.MCP-1 Recombinant 5 µg 24041V

TNF-α Capture TN3-19.12 Hamster Ig Purified 0.5 mg 23351DDetection Polyclonal Rabbit Ig Biotin 0.5 mg 23442DStandard Rec.TNF-α Recombinant 5 µg 25031V

Assay Diluent;Cat. No. 26411E

Volume: 500 ml

Assay Diluent contains animalserum in a buffered solution with0.15% ProClin-150 as preservative.Intended uses are to block ELISAplates, to dilute the recombinantStandards and samples, and toprepare Working Detector for theBD OptEIA™ Sets ELISA Reagents.

GENES

CELLS PROTEINS

BD Pharmingen’s extensive portfolioof matched antibody ELISA pairsand protein standards for ratcytokines and chemokines offer theresearcher:

• Flexible assay development• Lower cost of ELISA analysis

per sample• Reagent use for other applications

such as western blotting

Matched Antibody ELISA Pairs and Protein Standards

OptEIA™ Sets General ELISA Reagents

TMB Substrate Reagent Set; Cat. No. 2642KK

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

Substrate Reagent A containshydrogen peroxide in a bufferedsolution. Substrate Reagent B contains 3, 3’, 5, 5’-tetramethylben-zidine in organic solvent. Whenmixed together, TMB substrate solution reacts with peroxidase-labeled conjugates to develop a bluecolor. After addition of stoppingsolution (1M phosphoric or 2N sulfuric acid), the color changesfrom blue to yellow and themicrowells are read at 450 nm.


Cell Biology: Apoptosis and Neurobiology

GENES

CELLS PROTEINS

ApoptosisApoptosis, a form of programmedcell death, is a normal physiologicalprocess which occurs during normalembryonic development and in themaintenance of tissue homeostasis.Inappropriate induction of apoptosis has broad ranging pathologic implications and as aresult, the apoptosis field is ofincreasing scientific interest, withthousands of new publicationsoccurring every year. BD PharMingenoffers a number of reagentsdesigned to identify, quantitate,and characterize apoptosis.

NeurobiologyIn addition to our apoptosis line ofproducts, we have a number of antibodies for the study of cellularand neurobiological systems.BD PharMingen offers a wide variety of antibodies and otherreagents for neurobiologicalresearch in the rat, including antibodies to neurotransmitters andtheir receptors, glutamate receptorsand structural proteins, tumorsuppressors, oncogenes, cell cycle-related proteins, and a wide varietyof other intracellular proteins.

11697

66

55

3631

21

1 2

kD

143 4 5

p45

p33Caspase-1

97

55

31

Caspase-2

116

66

36

kD

1 2 3

Figure 72. Western blot analysis of caspase-1.HeLa human cervical carcinoma (lane 1), 293adenovirus-transformed human embryonic kid-ney (lane 2), Jurkat T cell (lane 3), BALB/c thy-mocytes (lane 4) and DC-3 SV40-transformed,rat ovarian granulosa cells (lane 5) probed withanti-caspase-1 (clone B24-2, Cat. No. 66441A).Caspase-1 is identified as 45 kD (precursor) and33 kD (intermediate) bands. In rat species, cloneB24-2 primarily detects the 45 kD form of theprotein.

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

ppRb&

pRb

RatREF F111

+- +-

Figure 74. Western blot analysis of Rb, clone G3-245 (Cat. No. 14001A) in cell cycling (+) and qui-escent (-) rat cell cultures. Most highly phosphorylated species of Rb are seen in cyclingcompared to quiescent cultures. REF, primary ratembryo fibroblasts. F111, established rat fibrob-lasts. ppRb and pRb represent different phos-phorylation states of Rb. The higher molecularweight bands (116 kD) are more highly phos-phorylated than the lower bands (110 kD).

Figure 75. Analysis of p53 expression in SV40-transformed rat ovarian cells (DC3) and rat thymo-cytes. Cells were analyzed by flow cytometry usinga p53-PE antibody (G59-12) (green histogram) oran IgG1-PE isotype control (MOPC-21) (open histogram), or by western blot analysis using apanel 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 detectedin DC3 cells but not in rat thymocytes. The PE-conjugated p53 and IgG1 antibodies are sold as aset (Cat. No. 6733KK).

41 2 3 1010 10 10100

016

012

080

2020

0

Cel

l Nu

mb

er

p53-PE (G59-12)

DC3

116 97 66 55

36 31

21

DC3

kD

p53

G59

-12

IgG

1

PAb

122

PAb

240

080

6040

20C

ell N

um

ber

41 2 3 1010 10 10100

100

p53-PE (G59-12)

Rat Thymocytes

116 97 66 55

36 31

21

Rat Thymocytes

kD G59

-12

IgG

1

PAb

122

PAb

240


Cell Biology: Apoptosis and Neurobiology, continued

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

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

Figure 78. Frozen rat brain tis-sue stained for 5-HT2AR (cloneG186-1117, Cat. No. 60231A).

5-HT2AR

1 2 3

Figure 79. Western blot analysis of5-HT2AR in rat brain synaptic mambranes.Blots were incubated with either G186-1117. (lane 1), G186-1117, absorbed withrecombinant GST-5-HT2AR fusion protein(lane 2), or G186-1117 absorbed with anirrelevant (GST-p16) fusion protein (lane 3).The antibody recognizes 2-HT2AR as an~55 kD band.

200

116976655

3631

2114

5-HT2CR

1 2

kD Figure 81. Western blot analysis of 5-HT2CRin rat brain synaptic plasma membrane.(Lane 1), clone A4-2 (Cat. No. 60541A) anIgG1 isotype control. (Lane 2), the antibodyrecognizes 5-HT2CR as e 55kDa band.

A

B

Figure 82. Formalin-fixed frozenadult rat brain sections (cerebel-lum) stained with the antiGABAb receptor (Cat. No.60696E). Arrow indicatesGABAb Receptor immunoreac-tivity. Strong immunostaining isseen in the cell bodies and den-drites of Purkinje cells. B is ahigher magnification of A.

Figure 83. Immunoprecipitation (IP) oflarge T antigen in SV40-transformed ratovarian cells (DC3). DC3 cell lysates wereimmunoprecipitated with antibodies top53 (G59-12, Cat. no. 14211A), SV40 large Tantigen (PAb 100, Cat. no. 14141A) or withan IgG1 isotype control (clone A85-1, Cat.no. 02231D). Proteins were separated bySDS-PAGE and then western blotted (WB)with an antibody recognizing large T anti-gen (PAb 101, Cat. no. 14111A). The resultsshow that SV40 large T could be immuno-precipitated with anti-large T antibody(PAb 100) or co-immunoprecipitated withan anti-p53 antibody (G59-12).

116

200

976655

3631

21

kD

IP:Ig

G1p5

3 (G

59-1

2)

SV40 (P

Ab 10

0)

SV40 (PAb 101)

SV40 Large T

Heavy chain

Light chain

WB:

Figure 76. Acetone-fixed, frozentissue section of rat brainstained for somatostatin (cloneYC7,Cat. No. 60132A).


GENES

CELLS PROTEINS

Antibodies to Rat Cell Biology Markers

Specificity Clone Application Cat. No. Format SizeAKT polyclonal WB 68601N Se 100 µlAKT (P-Ser473) polyclonal WB, IP 68591N Se 100 µlAndrogen G122-25 FC, IHCF, IHCP 15051A Pu 0.1 mgReceptorAndrogen G122-77 IP, IHCF 15071A Pu 0.1 mgReceptorDBP 10 WB 14681A Pu 0.1 mgE2F-1 KH95-E2F WB, IP 14971A Pu 0.1 mgFKBP-12 2C1-87 WB, IHC 13521A Pu 0.1 mgJun G56-206 WB 13411A Pu 0.1 mgJun IKK-γ WB 68341A Pu 0.1 mgKu-70/80 162 IP, IF, FC, ELISA 66181A Pu 0.1 mgL1 Protein CAMVIR WB 14481A Pu 0.1 mg(HPV type 16)Latent TGFβ-1 BP polyclonal IP, WB, IHCF, IHCP 15866E Se 100 µlMyoD M0Ab5.8A IP, WB, IHCF 13941A Pu 0.1 mgPLC-γ1 polyclonal IP, WB 15526E Se 100 µlPLC-γ2 polyclonal IP, WB 15996E Se 100 µlPKC MC5 IP, WB, IHCF, IF 14871A Pu 0.1 mgSC35 alphaSC35 IP, WB, IF 65201A Pu 0.1 mgSos1 polyclonal WB, IHC 15191B Lyo 0.2 mgSp1 1C6 WB 13931A Pu 0.1 mgStat1 IF9A7 WB, IP, IF 15221A Pu 0.1 mgTCP-1 22B IP, WB, IF 13321A Pu 0.1 mgTCP-1 84A IP, WB, IF 13311A Pu 0.1 mgTCP-1 91A IP, WB, IF 13331A Pu 0.1 mg

Antibodies to Rat Tumor Suppressors and Oncogene Markers

Specificity Clone Application Cat. No. Format SizeMOM2 SMP14 IP, WB, IHCF, 65101A Pu 0.1 mg

IHCPp53 G59-12 WB, IP, IHCF, 14211A Pu 0.1 mg

IHCPp53 PAb 122 WB, IP, IF 14091A Pu 0.1 mgp53 PAb 240 WB, IP, IHCF 14461A Pu 0.1 mgp53 FITC SET G59-12, FC 6732KK FITC 100 tests

MOPC-21p53 PE SET G59-12, FC 6733KK PE 100 tests

MOPC-21p53 PE SET DO-7,27-35 FC 6680KK PE 100 testsRb G3-245 WB, IP, FC, IF, 14001A Pu 0.1 mg

IHCF, IHCP, GSRb Cy-Chrome™ Set G3-245, FC 6805KK Cy-Chrome™ 100 tests

MOPC-21Rb FITC Set G3-245, FC 6684KK FITC 100 tests

MOPC-21RB PE Set G3-245, FC 6685KK PE 100 tests

MOPC-21SV40 PAb-100 IP, IF, IHCF 14141A Pu 0.1 mgLarge T AntigenSV40 PAb-101 WB, IP, IF, IHCF 14111A Pu 0.1 mgLarge T Antigen

Antibodies to Rat Cell Cycle Markers

Specificity Clone Application Cat. No. Format SizeCdK4 DCS-35 WB,IP 68941A Pu 0.1 mgCyclin A BF683 WB, IP 14531A Pu 0.1 mg

WB, IP 14531C Pu 0.25 mgFC 1370KK FITC Set 100 tests

Cyclin D1 G124-3216 WB, IP 14561A Pu 0.1 mgWB, IP 14561C Pu 0.1 mgFC 1372KK FITC Set 100 tests

Cyclin D1 DCS-6 WB, IP 66271A Pu 0.1 mg


Antibodies to Rat Cell Cycle Markers, continued

Specificity Clone Application Cat. No. Format SizeCyclin D3 G107-565 WB, IP 14781A Pu 0.1 mg

WB, IP 14781C Pu 0.25 mgFC 1374KK FITC Set 100 tests

Cyclins D1/D2/D3 G124-259 WB, IP 14841A Pu 0.1 mgWB, IP 14841C Pu 0.25 mgFC 1375KK FITC Set 100 tests

Antibodies to Rat Neurobiology Markers

Specificity Clone Application Cat. No. Format Size14-3-3 8C3 WB, IP 68291A Pu 0.1 mgDopamine DBH-41 WB 60101A Pu 0.1 mgβ-HydroxylaseGABA B Receptor polyclonal WB, IHCF 60696E Se 100 µlGFAP (cocktail) 4A11, WB, IF, 60341D Pu 0.5 mg

1B4,2E1 IHCF, IHCPGFAP 4A11 WB, IF, IHC 60311D Pu 0.5 mgGFAP 1B4 WB, IF, IHC 60321D Pu 0.5 mgGFAP 20 WB, IF, IHC 60331D Pu 0.5 mgGlutamate R polyclonal WB, IP, IHCF 60656N Pu 0.05 mg(GluR2,R3)Glutamate R polyclonal WB, IP 60666N Pu 0.05 mg(GluR4)Glutamate R 3A11 WB, IF, IHC 60011A Pu 0.1 mg(GluR 5,6,7)Glutamate R 54.1 WB, IHC 60021A Pu 0.1 mg(NMDAR1)MAP2 (a+b) Ap20 WB, IHCF 60171A Pu 0.1 mgMASH1 24B7.2D11 WB 68271A Pu 0.1 mgMyelin Myelin BP WB 69151A Pu 0.1 mgBasic ProteinMyogenin F5D WB, GS, IF, 65121A Pu 0.1 mg

IHCF, IHCPNF-YA YA-1a WB, GS 65131A Pu 0.1 mgNCAM 12F8 WB, IHCF 60221A Pu 0.1 mgPan N-CAM 12F11 WB, IHCF 60201A Pu 0.1 mgPan N-CAM N-CAM 13 WB, IHCF 60211A Pu 0.1 mgβ Nerve NGF30 WB, IHCF 60111A Pu 0.1 mgGrowth FactorNestin Rat 401 WB, FC, IF, IHCF 60051A Pu 0.1 mg(Glutamate Receptor)NMDAR1 54.1 WB 60021A Pu 0.1 mgNMDAR1 (C2') polyclonal WB 69041G Se .25 mg(Glutamate Receptor)NMDAR1 (C1) polyclonal WB 69021G Se .25 mg(Glutamate Receptor)NMDAR1 (C2) polyclonal WB 69031G Se .25 mg(Glutamate Receptor)NMDAR1 (N1) polyclonal WB 69051G Se .25 mg(Glutamate Receptor)Tau Tau-2 WB, IHCF, IHCP 60151A Pu 0.1 mg(Tau-2 Epitope)(k) Opiod polyclonal WB, IHCP 60711A Pu 0.1 mgReceptor (N-terminus)(k) Opiod polyclonal WB 60721A Pu 0.1 mgReceptor (internal region)(k) Opiod KA8 IP, IHC, EIA 60501A Pu 0.1 mgReceptor (N-terminus)Serotonin YC5/45 IHCF 60121A Pu 0.1 mgSerotonin G186-1117 WB, IHCF 60231A Pu 0.1 mgReceptor (5-HT2AR)Serotonin A72-1 WB, IHCF 60531A Pu 0.1 mgReceptor (5-HT2BR)Serotonin A4-2 WB, IHCF 60541A Pu 0.1 mgReceptor (5-HT2CR)

GENES

CELLS PROTEINS


Antibodies to Rat Neurobiology Markers, continued

Specificity Clone Application Cat. No. Format SizeSomatostatin YC7 IHCF 60131A Pu 0.1 mgSubstance P NC1/34 RIA, IHCF 60091A Pu 0.1 mgTrkA polyclonal IHCF 68266E Se 100 µlTau Tau-5 WB, IHCF, IHCP 60161A Pu 0.1 mg(Tau-5 Epitope)β-Tubulin 5H1 WB, IF 60181A Pu 0.1 mgTRH,TYH, PH8 WB, IP, 60061D Pu 0.5 mgand PAH IHCF, IHCPHydroxylasesTyrosine TOH A1 IHCF 60081A Pu 0.1 mgHydroxylaseVAchT polyclonal IHCF 60596E Se 100 µl

Antibodies to Apoptosis Markers

Specificity Clone Application Cat. No. Format SizeBak polyclonal WB 65606E Se 100 µlBax 6A7 WB, IP 66241A Pu 0.1 mgBax polyclonal WB, IP, IHCF, IHCP 13686E Se 100 µlBcl-2 polyclonal WB, IP, IHCP 13456E Se 100 µlBcl-2 polyclonal WB, IP, IHCP 15616E Se 100 µlBcl-XL 2H12 WB 66461A Pu 0.1 mgBim polyclonal WB 68871N Se 100 µlE2F-1 KH95/E2F 14971A 14971A Pu 0.1 mgCaspase-1 B24-1 WB 66441A Pu 0.1 mgCaspase-2 G310-1248 WB 13951A Pu 0.1 mgCaspase-4 B25-1 WB 66171A Pu 0.1 mgCytochrome c 6H2.B4 IP 65971A Pu 0.1 mgCytochrome c 7H8.2C12 WB 65981A Pu 0.1 mgPARP 7D3-6 WB 66391A Pu 0.1 mgPARP C-210 WB, IF 65196E Asc 100 µlRaf URP26K IP, WB 13981A Pu 0.1 mgBak polyclonal WB, IHCP 65606E Pu 100 µl

GENES

CELLS PROTEINS


BD RiboQuant™ RibonucleaseProtection Assay (RPA) Systems

GENES

CELLS PROTEINSThe ribonuclease protection assay(RPA) is a highly sensitive and specific method to detect and quantitate RNA. The method isbased on the hybridization of targetRNA to 32P-labeled anti-sense RNAprobes that have beenin vitro-transcribed from a DNA template. RNase treatment follows,resulting in degradation of single-stranded RNA and excessprobe. The probe and target RNAare resolved by denaturing polyacrylamide gel electrophoresisand the "protected" probe is visual-ized using autoradiography or phosphorimaging equipment.

BD PharMingen’s Multi-ProbeTemplate Sets are composed ofa series of biologically related templates, each of distinct lengthand each representing a sequencein a distinct mRNA species.

Each template set is capable ofdetecting up to 11 unique genetranscripts in addition to twohousekeeping genes, L32 andGAPDH. Samples of total RNAfrom various sources analyzed fordistinct mRNA species usingBD PharMingen’s RiboQuant™Multi-Probe RibonucleaseProtection Assay System. Theautoradiograms show the probe setsnot treated with RNases (Lanes 1)along with the correspondingRNase-protected probes followinghybridization with yeast tRNA(2 mg) (Lanes 2) and selected ratRNA (Lanes 3, 4, 5). Note thateach undigested control probe band(Lanes 1) migrates more slowly thanits protected and digested counter-point (Lanes 3); this is due to thedigestion of the flanking sequencesin the probe that are not protectedby the target mRNA. (Figures 84-88)

Rat RiboQuant™ Products

Description Size Cat. No.BD RiboQuant™ RPA Starter Package 10 rxns 45024KBD RiboQuant™ In Vitro Transcription Kit 25 rxns 45004KBD RiboQuant™ RNase Protection Assay Kit 200 rxns 45014KRNA Isolation Kit 45520K

Rat Cytokine Template SetsrCK-1 Rat Cytokine Template Set 10 rxns 45027PrCK-2 Rat Cytokine Template Set 10 rxns 45630PrCK-3 Rat Cytokine Template Set 10 rxns 45631P

Rat Neurobiology Template SetsrNT-1 Rat Neurotrophin Template Set 45028P

Rat Apoptosis Template SetsrAPO-1 RiboQuant™ Rat Apoptosis 45601PTemplate Set


GENES

CELLS PROTEINS

1 2 3rAPO-1

L32

GAPDH

Pro

be

Yea

st t

RN

A

Rat

Co

ntr

ol R

NA

Fas

caspase-2

FasL

caspase-1

caspase-3

bcl-x L/S

bax

bcl-2

1 2 3 4rNT-1

L32

GAPDH

Pro

be

βNGFBDNF

GDNF

CNTF

NT3

NT4

Yea

st t

RN

A

Rat

Bra

in R

NA

(5 µ

g)

Rat

Bra

in R

NA

(15

µg)

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

FAS 435 406bcl-X (L) 393 363 (L)bcl-X (S) 393 325 (S)FASL 315 286caspase-1 282 253caspase-3 255 226caspase-2 231 202 (L)bax 210 181bcl-2 189 160caspase-2 231 137 (S)L32 141 112GAPDH 126 97

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

βNGF 351 322BDNF 315 286GDNF 282 253CNTF 255 226NT3 231 202NT4 213 184L32 141 112GAPDH 126 97

1 2 3 4 5rCK-1

L32

GAPDH

Pro

be

Yea

sttR

NA

IL-1αIL-1βTNFβ

IL-3

IL-4

IL-5

IL-6

IL-10

TNFα

IL-2

IFNγ

αCD3: + + +αCD28: - + +

CD

4

CD

4

CD

8

T-Cell

1 2 3

rCK-2

IL-1αIL-1βIL-1Ra

IL-12p40

IL-18

IL-6

IL-10

MIF

IFNγ

L32

GAPDH

Pro

be

Yea

st t

RN

AR

atC

on

tro

l RN

A

1 2 3 4

rCK-3

IFNβTNFβGM-CSF

TGFβ1

TGFβ3

TGFβ2

Ltβ

TNFα

MIF

IFNγ

L32

GAPDH

Pro

be

Yea

st t

RN

A

Rat

Co

ntr

ol R

NA

Rat

Bo

ne

Mar

row

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

IL-1α 432 403IL-1β 390 361TNFβ 351 322IL-3 315 286IL-4 285 256IL-5 255 226IL-6 231 202IL-10 210 181TNFa 189 160IL-2 171 142IFNg 156 127L32 141 112GAPDH 126 97


IL-1α 432 403IL-1β 390 361IL-1Ra 351 322IL-12p40 285 256IGIF/IL-18 255 226IL-6 231 202IL-10 210 181MIF 171 142IFNγ 158 127L32 141 112GAPDH 126 97


IFNβ 390 361TNFβ 351 322GM-CSF 324 295TGFβ1 285 256TGFβ3 255 226TGFβ2 231 202Ltβ 210 181TNFα 189 160MIF 171 142IFNγ 158 127L32 141 112GAPDH 126 97

Special thanks to Dr. Frans Kroese for histechnical contribution to the developmentof this brochure.

Figure 84

Figure 85

Figure 88

Figure 87Figure 86


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