BrdU Flow Kit

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1 BrdU FLOW KIT Kit Manual

2 FITC -anti-brdu Antibody (Kit Component. No L) Cytofix/Cytoperm Buffer (Kit Component. No. 2090KE) Perm/Wash Buffer (10x (Kit Component. No. 2091KE) Cytoperm Plus Buffer (Kit Component. No. 2356KC) 7-AAD (Kit Component. No. 2359KC) Kit Manual BrdU (Kit Component. No. 2420KC) DNase (Kit Component. No. 2358KC)

3 BD Pharmingen TM BrdU FLOW KIT MANUAL i

4 ii

5 1

6 Section 1 BD Pharmingen BrdU Flow Kit 2

7 Section 1 BrdU Flow Kit 3

8 Section 2 4

9 µ µ µ 5

10 µ µ µ µ µ µ µ µ µ µ µ 6

11 µ Section 3 BrdU BrdU Flow Kit A. BrdU µ µ 7

12 µ µ 8

13 B. BrdU Flow Kit µ µ µ µ µ 9

14 µ µ µ µ µ 10

15 Section 4 11

16 A. BrdU B. IL-10 CD8 C. 7AAD BrdU D. CD8 BrdU IL-10 µ 12

17 A. B. C. D. CD8(PE) CD8(PE) CD8(PE) CD8(PE) CD4 (FITC) B220 (APC) CD4 (FITC) B220 (APC) % Positive MFI Conventional BrdU Conventional BrdU Protocol Protocol Protocol Protocol CD4 + FITC CD8 + PE B220 + APC

18 10 4 R BrdU (FITC) R6 R R AAD µ 14

19 10 4 A. 40 Minutes Thymus B. 2 Hours C Hours BrdU (FITC) BrdU (FITC) BrdU (FITC) AAD AAD Bone Marrow A. 40 Minutes B. 2 Hours C AAD 4 Hours BrdU (FITC) BrdU (FITC) BrdU (FITC) AAD AAD AAD 15

20 Section 5 16

21 A1 A2 A3 A4 α 17

22 A1 A2 A3 A4 18

23 A1 A2 A3 A4 19

24 A1 A2 A3 A4 20

25 A1 A2 A3 A4 21

26 A1 A2 A3 A4 PMT B1 B2 B3 B4 α α α α α 22

27 Section 6 23

28 24

29 Section 7 µ Specificity Clone Isotype New Cat. No. Old Cat. No. Format Size Human Cytokines and Chemokines IL-1α 364-3B3-14 Mouse IgG A FITC 0.1 mg A PE 0.1 mg IL-2 MQ1-17H12 Rat IgG2a A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests A APC 0.1 mg IL-3 BVD3-1F9 Rat IgG A PE 0.1 mg IL-4 MP4-25D2 Rat IgG A FITC 0.1 mg A PE 0.1 mg A APC 0.1 mg IL-4 8D4-8 Mouse IgG A PE 0.1 mg X PE 100 Tests IL-5 TRFK5 Rat IgG A PE 0.1 mg X PE 100 Tests A APC 0.1 mg IL-5 JES1-39D10 Rat IgG2a A PE 0.1 mg X PE 100 Tests IL-6 MQ2-13A5 Rat IgG A FITC 0.1 mg A PE 0.1 mg IL-6 MQ2-6A3 Rat IgG2a A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests 25

30 Specificity Clone Isotype New Cat. No. Old Cat. No. Format Size Human Cytokines and Chemokines (Continued) IL-8 G265-8 Mouse IgG2b A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests IL-10 JES3-9D7 Rat IgG A PE 0.1 mg X PE 100 Tests IL-10 JES3-19F1 Rat IgG2a A PE 0.1 mg X PE 100 Tests A APC 0.1 mg IL-12 (p40/p70) C Mouse IgG A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests A APC 0.1 mg IL-12 (p70) 20C2 Rat IgG A PE 0.1 mg X PE 100 Tests IL-13 JES10-5A2 Rat IgG A PE 0.1 mg X PE 100 Tests IL Mouse IgG2a A PE 0.1 mg GM-CSF BVD2-21C11 Rat IgG2a A FITC 0.1 mg A PE 0.1 mg GRO 10G4 Mouse IgG A PE 0.1 mg IFN-γ B27 Mouse IgG A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests A APC 0.1 mg IFN-γ 4S.B3 Mouse IgG A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests IP-10 6D4/D6/G2 Mouse IgG2a A PE 0.1 mg MCP-1 5D3-F7 Mouse IgG A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests MCP-3 9H11 Mouse IgG A PE 0.1 mg MIG B8-11 Mouse IgG A PE 0.1 mg 26

31 Specificity Clone Isotype New Cat. No. Old Cat. No. Format Size Human Cytokines and Chemokines (Continued) MIP-1α 11A3 Mouse IgG2a A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests RANTES 2D5 Mouse IgG A PE 0.1 mg X PE 100 Tests Thioredoxin 2G11/TRX Mouse IgG A FITC 0.1 mg TNF-α MAb11 Mouse IgG A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests A APC 0.1 mg TNF-β Mouse IgG A PE 0.1 mg Specificity Clone Isotype New Cat. No. Old Cat. No. Format Size Mouse Cytokines and Chemokines IL-1α ALF-161 Hamster IgG A PE 0.1 mg IL-2 JES6-5H4 Rat IgG2b A FITC 0.1 mg A PE 0.1 mg A APC 0.1 mg IL-2 S4B6 Rat IgG2a A FITC 0.1 mg A PE 0.1 mg IL-3 MP2-8F8 Rat IgG A PE 0.1 mg IL-4 BVD4-1D11 Rat IgG2b A FITC 0.1 mg A PE 0.1 mg IL-4 11B11 Rat IgG A PE 0.1 mg A APC 0.1 mg IL-5 TRFK5 Rat IgG A PE 0.1 mg A APC 0.1 mg IL-6 MP5-20F3 Rat IgG A PE 0.1 mg IL-10 JES5-16E3 Rat IgG2b A FITC 0.1 mg A PE 0.1 mg A APC 0.1 mg IL-12 (p40/p70) C15.6 Rat IgG A PE 0.1 mg A APC 0.1 mg 27

32 Specificity Clone Isotype New Cat. No. Old Cat. No. Format Size Mouse Cytokines and Chemokines (Continued) IL-17 TC11-18H10 Rat IgG A PE 0.1 mg GM-CSF MP1-22E9 Rat IgG2a A FITC 0.1 mg A PE 0.1 mg IFN-γ XMG1.2 Rat IgG A FITC 0.1 mg A PE 0.1 mg A APC 0.1 mg MCP-1 2H5 Hamster IgG A PE 0.1 mg TNF-α MP6-XT22 Rat IgG A FITC 0.1 mg A PE 0.1 mg A APC 0.1 mg TNF-α TN Hamster IgG A PE 0.1 mg Specificity Clone Isotype New Cat. No. Old Cat. No. Format Size Rat Cytokines and Chemokines IL-4 OX-81 Mouse IgG A PE 0.1 mg IL-10 A5-4 Mouse IgG2b A PE 0.1 mg IFN-γ DB-1 Mouse IgG X FITC 100 Tests X PE 100 Tests GM-CSF B61-5 Mouse IgG A PE 0.1 mg MCP-1 2H5 Hamster IgG A PE 0.1 mg TNF-α TN Hamster IgG A PE 0.1 mg Pig Cytokines and Chemokines IFN-γ P2G10 Mouse IgG A PE 0.1 mg Recombinant proteins useful as specificity controls for intracellular cytokine flow cytometry Specificity New Cat. No. Old Cat. No. Format Size Human IL-1α V Standard 5 µg IL T Standard 10 µg IL T Standard 10 µg IL V Standard 5 µg IL V Standard 5 µg IL T Standard 10 µg IL P Standard 20 µg 28

33 Specificity New Cat. No. Old Cat. No. Format Size Human (Continued) IL V Standard 5 µg IL V Standard 5 µg IL V Standard 5 µg GM-CSF T Standard 10 µg MIP-1α T Standard 10 µg TNF-α T Standard 10 µg TNF-β T Standard 10 µg Specificity New Cat. No. Old Cat. No. Format Size Mouse IL P Standard 20µg L T Standard 10 µg IL T Standard 10 µg IL V Standard 5 µg IL V Standard 5 µg IL T Standard 10 µg GM-CSF T Standard 10 µg MCP V Standard 5 µg TNF-α T Standard 10 µg Rat IL V Standard 5 µg IL V Standard 5 µg GM-CSF V Standard 5 µg MCP V Standard 5 µg Intracellular Cytokine-Positive Control Cells Description Cytokines Expressed New Cat. No. Old Cat. No. Human HiCK 1 Positive for IL-2, IFN-γ, TNF-α Z HiCK 2 Positive for IL-3, IL-4, IL-10, IL-13, GM-CSF Z HiCK 3 Positive for IL-1α, IL-1β, IL-6, IL-12, TNF-α Z HiCK 4 Positive for IL-8, GRO, IP-10, MCP-1, Z MCP-3, MIG, MIP-1α, 29

34 Intracellular Cytokine-Positive Control Cells Description Cytokines Expressed New Cat. No. Old Cat. No. Mouse MiCK 1 Positive for IL-2, IFN-γ, TNF-α Z MiCK 2 Positive for IL-3, IL-4, IL-10, GM-CSF, TCA Z MiCK 3 Positive for IL-1α, IL-6, IL-12, MCP-1, TNF-α Z Rat RiCK 2 Positive for IL-4, IL-10, GM-CSF, IFN-γ, TNF-α Z Specificity Clone New Cat. No. Old Cat. No. Format Size Isotype Controls Mouse IgG1 MOPC A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests A APC 0.1 mg Mouse IgG2a G A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests Mouse IgG2b A FITC 0.1 mg A PE 0.1 mg Rat IgG1 R A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests A APC 0.1 mg Rat IgG2a R A FITC 0.1 mg A PE 0.1 mg X PE 100 Tests A APC 0.1 mg Rat IgG2b A A FITC 0.1 mg A PE 0.1 mg A APC 0.1 mg Hamster IgG G A FITC 0.1 mg A PE 0.1 mg 30

35 Intracellular Cytokine Flow Cytometry Kits and Reagents Description New Cat. No. Old Cat. No. Kits Cytofix/Cytoperm Kit KK Cytofix/Cytoperm Plus (with Golgi Stop) KK Cytofix/Cytoperm Plus (with GolgiPlug) KK Human Intracellular Cytokine Staining Starter Kit KK Mouse Intracellular Cytokine Staining Starter Kit KK BrdU Flow Kit (FITC) KK Description New Cat. No. Old Cat. No. Size Reagents/Buffers Cytofix/Cytoperm Buffer KZ 125 mls Perm/Wash Buffer (10X) KZ 100 mls GolgiStop (containing monensin) KZ 0.7 ml GolgiPlug (containing brefeldin A) KZ 1.0 ml Cytofix Buffer KZ 100 mls Pharmingen Stain Buffer (FBS) E 500 mls Pharmingen Stain Buffer (BSA) E 500 mls 31

36 Section 8 1. Sasaki, K., T. Murakami and M. Takahashi Flow cytometric analysis of cell proliferation kinetics using the anti-brdurd antibody. Gan To Kagaku Ryoho. 16: Miltenburger, H. G., G. Sachse and M. Schliermann S-phase cell detection with a monoclonal antibody. Dev. Biol. Stand. 66: Vanderlaan, M. and C. B. Thomas Characterization of monoclonal antibodies to bromodeoxyuridine. Cytometry. 6: Gratzner, H. G. and R. C. Leif An immunofluorescence method for monitoring DNA synthesis by flow cytometry. Cytometry. 1: Lacombe, F., F. Belloc, P. Bernard, M. R. Boisseau Evaluation of four methods of DNA distribution data analysis based on bromodeoxyuridine/dna bivariate data. Cytometry. 9: ean, P. N., F. Dolbeare, H. Gratzner, G. C. Rice and J. W. Gray Cell-cycle analysis using a monoclonal antibody to BrdUrd. Cell Tissue Kinet. 17: Toba, K., E. F. Winton and R. A. Bray Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation. Cytometry. 13: Sasaki, K., S. Adachi, T. Yamamoto, T. Murakami, K. Tanaka and M. Takahashi Effects of denaturation with HCl on the immunological staining of bromodeoxyuridine incorporated into DNA. Cytometry. 9: Lakhanpal, S., N. J. Gonchoroff, J. A. Katzmann and B. S. Handwerger A flow cytofluorometric double staining technique for simultaneous determination of human mononuclear cell surface phenotype and cell cycle phase. J. Immunol. Meth. 96: Houck, D. W. and M. R. Loken Simultaneous analysis of cell surface antigens, bromodeoxyuridine incorporation and DNA content. Cytometry. 6: Moran, R., Z. Darzynkiewicz, L. Staiano-Coico and M. R. Melamed Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step. J. Histochem. Cytochem. 33: Holm, M., M. Thomsen, M. Høyer and P. Hokland Optimization of a flow cytometric method for the simultaneous measurement of cell surface antigen, DNA content, and in vitro BrdUrd incorporation into normal and malignant hematopoietic cells. Cytometry. 32: Mehta, B. A. and V. C. Maino Simultaneous detection of DNA synthesis and cytokine production in staphylococcal enterotoxin B activated CD4 + T lymphocytes by flow cytometry. J. Immunol. Meth. 208: Endl, E., P. Steinbach, R. Knühel and F. Hofstäter Analysis of cell cycle-related Ki-67 and p120 expression by flow cytometric BrdUrd-Hoechst/7AAD and immunolabeling technique. Cytometry. 29: Dolbeare, F., H. Gratzner, M. G. Pallavicini and J. W. Gray Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc. Natl. Acad. Sci. USA. 80:

37 16. Penit, C in vivo thymocyte maturation. BrdU labeling of cycling thymocytes and phenotypic analysis of their progeny support the single lineage model. J. Immunol. 137: Thoman, M. L Early steps in T cell development are affected by aging. Cell. Immunol. 178: Tough, D. F., and J. Sprent Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 179: von Boehmer, H., and K. Hafen The life span of naive alpha/beta T cells in secondary lymphoid organs. J. Exp. Med. 177: Schittek, B., K. Rajewsky and I. Forster Dividing cells in bone marrow and spleen incorporate bromodeoxyuridine with high efficiency. Eur. J. Immunol. 21: Rocha, B., C. Penit, C. Baron, F. Vasseur, N. Dautigny, and A. A. Freitas Accumulation of bromodeoxyuridine-labeled cells in central and peripheral lymphoid organs: minimal estimates of production and turnover rates of mature lymphocytes. Eur. J. Immunol. 20: Westermann, J., S. Ronneberg, F. J. Fritz and R. Pabst Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs. Eur. J. Immunol. 19: Robey, E., D. Chang, A. Itano, D. Cado, H. Alexander, D. Lans, G. Weinmaster, and P. Salmon An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell. 87: Carayon, P. and A. Bord Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA. J. Immunol. Meth. 147: Gonchoroff, N. J., J. A. Katzmann, R. M. Currie, E. L. Evans, D. W. Houck, B. C. Kline, P. R. Greipp and M. R. Loken S-phase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment. J. Immunol. Meth. 93: Shapiro, H. M., Practical Flow Cytometry, 3rd Edition, Wiley-Liss, New York. 27. Gray, J. W. and Z. Darzynkiewicz, Eds., Techniques in Cell Cycle Analysis, Humana Press, Clifton, New Jersey. 33

38 34 Notes

39 Notes 35

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qxd BD Pharmingen TM BrdU Flow Kit FITC BrdU Flow Kit 559619 50 557891 4 50 APC BrdU Flow Kit 552598 50 557892 4 50 2005 Becton, Dickinson and Company. All rights reserved. BD Biosciences Becton, Dickinson