qxd

Size: px

Start display at page:

Download "qxd"

しらんつちかね
5 years ago
Views:

1 BD Pharmingen TM BrdU Flow Kit FITC BrdU Flow Kit APC BrdU Flow Kit

3 C Fluorochrome-conjugated Anti-BrdU Antibody: 1 BD Cytofix/CytopermTM Buffer: 1 BD Perm/WashTM Buffer 10x : 2 BD CytopermTM Plus Buffer: 1 7-AAD: 1 Kit Manual -80 C BrdU: 5 DNase:

4 BD Pharmingen TM BrdU Flow Kit BrdU BrdU Flow Kit A. BrdU B. BrdU Flow Kit

5 Bromodeoxyuridine BrdU DNA BrdU DNA S DNA DNA 1-4 BrdU anti-brdu BrdU 7-amino-actinomycin D 7-AAD DNA BrdU 2 7-AAD G0/1 S G2/M DNA BrdU 5, 6 BrdU BrdU BrdU in vitro in vivo BD Pharmingen TM BrdU Flow Kit BrdU BrdU Flow Kit DNA 7-11 BrdU Flow Kit DNA BrdU BrdU Flow Kit in vitro DNA T IL-2 BD Pharmingen BrdU Flow Kit BrdU BrdU BD Pharmingen TM BrdU In-Situ Kit

6 BD Pharmingen TM BrdU Flow Kit BD Pharmingen TM BrdU Flow Kit 1 3 BrdU BrdU Option #1 Option #2 BD Pharmingen BrdU Flow Kit 6

7 BD Pharmingen TM BrdU Flow Kit option no. 1 4 C Staining Buffer in vitro in vivo BrdU BD Cytofix/Cytoperm Buffer option no. 2 Staining Buffer -80 C 1 BD Cytoperm Plus Buffer Staining Buffer BD Cytofix/Cytoperm Buffer DNase BrdU 2 BrdU 3 7-AAD DNA 4 Staining Buffer 1. 10%dimethyl suifoxide DMSO 90% FBS 2. DNase DNase Perm/Wash Buffer Total DNA 7-AAD FL3 7

8 1 Fluorochrome- anti-brdu Antibody 4 C 1 BD Cytofix/Cytoperm Buffer 4 C 1 BD Perm/Wash Buffer 10x 4 C 2 BD Cytoperm Plus Buffer 4 C 1 BrdU 10 mg/ml -80 C 5 Dnase -80 C 5 7-AAD 4 C 1 1 Dulbecco PBS DPBS BD Perm/Wash Buffer Fluorochrome-conjugated anti-brdu antibody BrdU 50 µl / FITC BrdU Flow Kit FITC BrdU 50 µl APC BrdU Flow Kit APC BrdU 50 µl 1 BD Perm/Wash Buffer µl BrdU 4 C BD Cytofix/Cytoperm TM Buffer BD Cytofix/Cytoperm Buffer 25 ml BD Cytofix/Cytoperm Buffer BD Cytofix/Cytoperm Buffer 4 C BD Perm/Wash Buffer BD Perm/Wash Buffer ml BD Perm/Wash Buffer Perm/Wash Buffer 4 10 Perm/Wash Buffer BD Perm/Wash Buffer 10 perm/wash Buffer 0.45 µm BD Perm/Wash Buffer 1 8

9 BD Cytoperm TM Plus Buffer BD Cytoperm Plus Buffer BrdU Flow Kit µl/ BD Cytoperm Plus Buffer 10 ml 1 4 C BD Cytoperm Plus Buffer BrdU 1 DPBS 10 mg/ml BrdU 32.5 mm 0.5 ml BrdU 0.22 µm 1 mm in vitro in vivo 11 in vivo in vitro 10 mg/ml BrdU 31 µl 1 ml 1 x DPBS 32 1 mm 1 mm 10 µl 1 ml 10 µm BrdU BrdU 5-80 C BrdU 4 4 DNase 1 DPBS 1 mg/ml DNase 300 µl 10 DNase µl 1 DPBS 300 µg/ml DNase 10 1mg/mL DNase 30 µl/ 1 mg/ml DNase -80 DNase 1 mg DNase/mL µl µg DNase 37 DNase AAD 7-amino-actinomycin D 7-AAD DNA 10 6 / 20 µl 7-AAD 7-AAD 1 4 C Staining Buffer 1 DPBS 3% 0.09% BD Pharmingen TM Stain Buffer FBS DPBS 1 KCI KH 2 PO 4 NaCl Na 2 HPO 4 7H 2 O ph g 0.2 g 8.0 g 2.16 g 9

10 BD Cytofix/Cytoperm TM Buffer 4% R40 R43 S2 S13 S36/37 S46 S52 BD Cytoperm TM Plus Buffer 10% R20/21/22 S25 S26 S28 36 S60 BrdU 1% R20/21/22 S9 S28 S36/37/39 S60 0.1% 10

11 BrdU BrdU Flow Kit BrdU BrdU in vitro in vitro BrdU BrdU 10 µm 1 ml 1mM BrdU 10 µl 15, 16 BrdU BrdU in vitro 1 ml 10 µl BrdU 1 DPBS 1 mm BrdU /ml CTLL BrdU BrdU BrdU in vivo in vivo BrdU BrdU BrdU BD Biosciences Pharmingen No. 1 BrdU 1 DPBS BrdU 10 mg/ml in vivo BrdU µl 1 2 mg 17, 19, 21 BrdU 1 No. 2 BrdU BrdU 0.8 mg/ml BrdU 18, 23 BrdU 14 BrdU 21 BrdU 9 18 BrdU

12 BrdU Flow Kit 1. a. BrdU 10 6 /50 µl Staining Buffer b. Staining Buffer BD Pharmingen TM Stain Buffer FBS µl c. 15 d. 1 ml Staining Buffer xg 5 2. BD Cytofix/Cytoperm Buffer a. 100 µl BD Cytofix/Cytoperm Buffer b c. 1 ml 1 BD Perm/Wash Buffer 1 1d 3. BD Cytoperm Plus Buffer a. 100 µl BD Cytoperm Plus Buffer b. 10 c. 1 ml 1 BD Perm/Wash Buffer 1 2c 4. a. 100 µl BD Cytofix/Cytoperm Buffer b. 5 c. 1 ml 1 BD Perm/Wash Buffer 1 2c 5. 24, 25 DNase BrdU a. DNase 100 µl DPBS 300 µg/ml DNase 30 µg b c. 1 ml 1 BD Perm/Wash Buffer 1 2c 6. BrdU a. BrdU / 50 µl BD Perm/Wash Buffer b. 20 c. 1 ml 1 BD Perm/Wash Buffer 1 2c 7. DNA DNA 8 a. 20 µl 7-AAD 8. a. 1 ml Staining Buffer b. 400 /

13 488 nm fluorescein isothiocyanate FITC FL1 phycoerythrin PE FL2 7-AAD FL3 FSC SSC allophycocyanin APC BD FACSCalibur TM DNA 7-AAD BD Pharmingen FITC BrdU Flow Kit A 7-AAD vs. FITC anti-brdu BrdU S DNA Quadrant Marker 27% BrdU B APC anti-cd8 vs. FITC anti-brdu BrdU CD8 24% C APC anti-cd8 vs. PE anti-il-10 IL-10 CD8 CD8 34% IL-10 D PE anti-il-10 vs. FITC anti-brdu BrdU IL-10 38% 1. DNA / IL-10 BALB/c in vitro PMA µm BrdU FITC anti-brdu FL1 PE anti-il-10 FL2 7-AAD FL3 APC anti-cd-8 FL4 2 13

14 2. BrdU Flow Kit FITC anti-cd-4 PE anti-cd8 APC anti-b220 BD Cytofix/Cytoperm TM Buffer A B BD Pharmingen BrdU C D 2 A C CD4 FITC vs. CD8 PE B D B220 APC vs. CD8 PE CD4 CD8 B220 MFl 2 2. BrdU %positive MFI BrdU BrdU CD4+ FITC CD8+ PE B220+ APC BrdU BrdU 2 BrdU BrdU 14

15 3. BrdU DNA A D10.G4.1 BrdU FITC anti-brdu DNA 7-AAD D10.G µm BrdU 30 DNA Total DNA BrdU 7-AAD vs. BrdU BrdU Flow Kit Sub-G0/Gl R6 5.6% G0/G1 R3 38.6% S R4 38.6% G2+M R5 14.4% DNA 5, 6 7-AAD X B BrdU APC anti-brdu Total DNA 7-AAD PBMC CD3 10 µg/ml HIT3a CD28 2 µg/ml CD ng/ml IL ng/ml IL IL-2 IL-4 3 PMA Sigma P ng/ml Sigma I ng/ml µm BrdU 3A % 4 S 23.5% 3 G0/G1 64.3% 5 G2+M 6.1% 15

4. in vivo BrdU C57BL/6 BrdU 1 mg 40 2 4 BrdU 7AAD A BrdU 40

16 4. in vivo BrdU C57BL/6 BrdU 1 mg BrdU 7AAD A BrdU 40 BrdU/7AAD B 2 BrdU G0/G1 7AAD BrdU C 4 G0/Gl BrdU BrdU/7AAD 16

17 PMT FL2-%FL1 FL2-%FL3 FL3-%FL2 FL2-% FL BrdU 5 10 BD Pharmingen TM FITC BrdU Flow Kit BD Pharmingen TM APC BrdU Flow Kit , 27 Howard M. Shapiro Practical Flow Cytometry 3rd Edition, Wiley-Liss, New York 17

18 FITC BrdU 5. FITC BrdU Flow Kit T in vitro PMA 45 BrdU BrdU Flow Kit DNase FITC anti-brdu FL1 7-ADD FL3 PE lg FL2 PE IL-10 PE TNF 5.A1 A4 PMT BD FACSComp TM Bead Lyse/Wash 5.B1 B2 BD CellQuest TM 5.Al A4 PMT SSC vs. FSC 5.A1 5.B2 FL3 7-AAD DNA 5.B2 7-AAD FL3 5.A3 G0/G1 3 MFI FL3 PMT FL1 FL2 PMT

19 6. FITC FL1 6.A1 6.A2 A4 FITC anti-brdu + FL1 FL2-%FL1 5 FL2-%FL1 FL2 6.A2 X PE Ig PE FL2 19

20 7. 7-ADD FL3 7-ADD+ FL3 FL2-%FL3 5 7.A2 FL2 1 MFI 10 FL2-%FL3 PE Ig PE FL2 20

21 8. PE FL2 PE anti-il-10 FITC anti-brdu 7-AAD 5 PE lg PMT PE anti-il-10 8.A1 A4 21

22 9. PE FL2 FL2-%FL3 1 FL3-%FL2 2 PE IL-10 8 FL2- %FL3 7-AAD FL3 FL2 1 IL-10 FL2 1 MFl 10 FL3-%FL2 8.B1 9.B1 8.A4 2 PE anti-il- 10 FL2 22

23 10. PE FL2 5 FITC anti-brdu 7-AAD PE anti-tnf 10.A2 A4 PE anti-il-10 9 PE anti-tnf TNF FL2 PE IL-10 9.A2 10.A2 PE FL2 TNF 10.A2 A4 PE FL2 7-AAD EL3 TNF 2 FL2 PMT FL2 EL3-%FL2 FL2-%FL B2 B4 PMT FL2-%FL1 FL1-%FL2 23

24 APC BrdU Instrument Set up 11. anti-brdu APC 5 FITC anti-brdu FITC PE 7-AAD APC anti-brdu APC anti-brdu 11.A1 SSC vs. FSC FL3 7-AAD DNA FL B2 11.A4 A6 FL4 BrdU C1 11.A4 G0/G FL3 PMT 7AAD 24

25 12. PE FL3 FL4 PMT FL2 PE FL4 FL4-%FL2 FL2-%FL3 FL2 FITC PE FL2-%FL A2 A3 A5 12.A2 A3 A5 FL2 7-AAD 25

13. PE FL2 PE FL2 FL2 FL3 PE anti-ter-119 553673

26 13. PE FL2 PE FL2 FL2 FL3 PE anti-ter AAD APC anti-brdu FITC PE PE Y FL3-%FL2 BrdU/7AAD 14 26

27 14. PE FL2 14.A3 13.A3 FL3-%FL2 PE PE G0/G1 S G2/M APC anti-brdu 7AAD FL3 vs. FL4 14.A4 27

15. FITC FL2 FITC anti-cd11b 557396 PE anti-ter-119 553673 7AAD APC anti-brdu FITC FL2 FL2- %FL1

28 15. FITC FL2 FITC anti-cd11b PE anti-ter AAD APC anti-brdu FITC FL2 FL2- %FL1 PE PE FITC vs. PE PE FL2-%FL3 FL3-%FL2 7AAD FL3 G0/G FL3 PMT FL2-%FL3 PE vs. 7AAD FITC 28

29 BrdU BD Pharmingen BrdU Flow Kit APC PE BD Pharmingen BrdU Flow Kit BD Pharmingen BrdU Flow Kit BD Pharmingen TM BrdU Flow Kit FACSCalibur 4 FACSCalibur TM 4 FL1 FL2 FL3 FL / 4 BrdU BD Pharmingen TM BrdU In-situ Detection Kit BrdU BrdU BrdU BD Pharmingen TM BrdU In-situ Detection Kit BrdU BD Retrievagen A BrdU BrdU 29

30 / 100 PE 20 µl/ Description Clone Isotype Format Size Cat. No. Human Cytokines and Chemokines IL-1α 364-3B3-14 Mouse IgG1, κ PE 0.1 mg IL-2 MQ1-17H12 Rat IgG 2a FITC 0.1 mg PE 0.1 mg PE 100 Tests APC 0.1 mg IL-3 BVD3-1F9 Rat IgG 1 PE 0.1 mg IL-4 MP4-25D2 Rat IgG 1 FITC 0.1 mg PE 0.1 mg APC 0.1 mg Alexa Fluor Tests Alexa Fluor Tests IL-4 8D4-8 Mouse IgG 1 PE 0.1 mg PE 100 Tests IL-5 TRFK5 Rat IgG 1 PE 0.1 mg APC 0.1 mg IL-5 JES1-39D10 Rat IgG 2a PE 0.1 mg PE 100 Tests IL-6 MQ2-13A5 Rat IgG 1 FITC 0.1 mg PE 0.1 mg IL-6 MQ2-6A3 Rat IgG 2a FITC 0.1 mg PE 0.1 mg PE 100 Tests GM-CSF BVD2-21C11 Rat IgG 2a PE 0.1 mg GRO 10G4.1 Mouse IgG 1, κ PE 0.1 mg IFN-γ B27 Mouse IgG 1 FITC 0.1 mg PE 0.1 mg PE 100 Tests APC 0.1 mg Alexa Fluor Tests Alexa Fluor Tests IFN-γ 4S.B3 Mouse IgG 1, κ FITC 0.1 mg PE 0.1 mg PE 100 Tests IL-8 G265-8 Mouse IgG 2b FITC 0.1 mg PE 0.1 mg IL-10 JES3-9D7 Rat IgG 1 PE 0.1 mg PE 100 Tests Alexa Fluor is a registered trademark of Molecular Probes, Inc., Eugene, Or. 30

31 Description Clone Isotype Format Size Cat. No. Human Cytokines and Chemokines (Continued) IL-10 JES3-19F1 Rat IgG 2a PE 0.1 mg PE 100 Tests APC 0.1 mg IL-12 (p40/p70) C11.5 Mouse IgG 1 FITC 0.1 mg PE 0.1 mg PE 100 Tests APC 0.1 mg IL-12 (p70) 20C2 Rat IgG 1, κ PE 0.1 mg PE 100 Tests IL-13 JES10-5A2 Rat IgG 1 PE 0.1 mg PE 100 Tests IL Mouse IgG 2a, κ PE 0.1 mg IP-10 6D4/D6/G2 Mouse IgG 2a, κ PE 0.1 mg MCP-1 5D3-F7 Mouse IgG 1 PE 0.1 mg PE 100 Tests MCP-3 9H11 Mouse IgG 1 PE 0.1 mg MIG B8-11 Mouse IgG 1, κ PE 0.1 mg MIP-1 11A3 Mouse IgG 2a, κ PE 0.1 mg RANTES 2D5 Mouse IgG 1 PE 0.1 mg PE 100 Tests Thioredoxin 2G11/TRX Mouse IgG 1 FITC 0.1 mg TNF MAb11 Mouse IgG 1 FITC 0.1 mg PE 0.1 mg PE 100 Tests APC 0.1 mg LT-α (TNF-β) Mouse IgG 1 PE 0.1 mg Mouse Cytokines and Chemokines IL-1 ALF-161 Hamster IgG PE 0.1 mg IL-2 JES6-5H4 Rat IgG 2b FITC 0.1 mg PE 0.1 mg APC 0.1 mg IL-3 MP2-8F8 Rat IgG 1 PE 0.1 mg IL-4 BVD4-1D11 Rat IgG 2b PE 0.1 mg IL-4 11B11 Rat IgG 1 PE 0.1 mg APC 0.1 mg IL-5 TRFK5 Rat IgG 1 PE 0.1 mg APC 0.1 mg IL-6 MP5-20F3 Rat IgG 1 PE 0.1 mg IL-10 JES5-16E3 Rat IgG 2b FITC 0.1 mg PE 0.1 mg APC 0.1 mg IL-12 (p40/p70) C15.6 Rat IgG 1 PE 0.1 mg APC 0.1 mg IL-17 TC11-18H10 Rat IgG 1, κ PE 0.1 mg GM-CSF MP1-22E9 Rat IgG 2a PE 0.1 mg IFN- XMG1.2 Rat IgG 1 FITC 0.1 mg PE 0.1 mg APC 0.1 mg MCP-1 2H5 Hamster IgG, κ PE 0.1 mg

32 Description Clone Isotype Format Size Cat. No. Mouse Cytokines and Chemokines (Continued) TNF MP6-XT22 Rat IgG 1 FITC 0.1 mg PE 0.1 mg APC 0.1 mg TNF TN Hamster IgG PE 0.1 mg Rat Cytokines and Chemokines IL-4 OX-81 Mouse IgG 1, κ PE 0.1 mg IL-10 A5-4 Mouse IgG 2b PE 0.1 mg IFN-g DB-1 Mouse IgG 1, κ FITC 100 Tests PE 100 Tests GM-CS B61-5 Mouse IgG 1 PE 0.1 mg MCP-1 2H5 Hamster IgG, κ PE 0.1 mg TNF TN Hamster IgG PE 0.1 mg Pig Cytokines and Chemokines IFN-γ P2G10 Mouse IgG 1 PE 0.1 mg

33 Recombinant proteins useful as specificity controls for intracellular cytokine flow cytometry Description Format Size Cat. No. Human IL-2 Standard 10 µg IL-3 Standard 10 µg IL-4 Standard 5 µg IL-5 Standard 5 µg IL-6 Standard 10 µg IL-8 Standard 20 µg IL-10 Standard 5 µg IL-16 Standard 5 µg GM-CSF Standard 10 µg MIP-1 Standard 10 µg TNF Standard 10 µg LT-a(TNF-b) Standard 10 µg Mouse IL-2 Standard 20 µg IL-3 Standard 10 µg IL-4 Standard 10 µg IL-5 Standard 5 µg IL-6 Standard 5 µg IL-10 Standard 10 µg GM-CSF Standard 10 µg MCP-1 Standard 5 µg TNF Standard 10 µg Rat IL-4 Standard 5 µg IL-10 Standard 5 µg GM-CSF Standard 5 µg MCP-1 Standard 5 µg

34 Intracellular Cytokine-Positive Control Cells Description Cytokines Expressed Cat. No. Human HiCK 1 Positive for IL-2, IFN-γ, TNF HiCK 2 Positive for IL-3, IL-4, IL-10, IL-13, GM-CSF HiCK 3 Positive for IL-1, IL-1, IL-6, IL-12, TNF HiCK 4 Positive for IL-8, GRO-, IP-10, MCP-1, MCP-3, MIG, MIP Mouse MiCK 1 Positive for IL-2, IFN-γ, TNF MiCK 2 Positive for IL-3, IL-4, IL-10, GM-CSF, TCA MiCK 3 Positive for IL-1, IL-6, IL-12. MCP-1, TNF Rat RiCK 2 Positive for IL-4, IL-10, GM-CSF, IFN-γ, TNF Description Clone Format Size Cat. No. Isotype Controls Mouse IgG1, MOPC-21 FITC 0.1 mg PE 0.1 mg PE 100 Tests APC 0.1 mg Mouse IgG2a, G FITC 0.1 mg PE 0.1 mg PE 100 Tests Mouse IgG2b, FITC 0.1 mg PE 0.1 mg Rat IgG1 R3-34 FITC 0.1 mg PE 0.1 mg PE 100 Tests APC 0.1 mg Rat IgG2a, R35-95 FITC 0.1 mg PE 0.1 mg PE 100 Tests APC 0.1 mg Rat IgG2b A95-1 FITC 0.1 mg PE 0.1 mg APC 0.1 mg Hamster IgG G PE 0.1 mg

35 Intracellular Cytokine Flow Cytometry Kits and Reagents Description Kits Cat. No. BD Cytofix/Cytoperm Kit BD Cytofix/Cytoperm Kit (with BD GolgiStop) BD Cytofix/Cytoperm Kit (with BD GolgiPlug) Human Intracellular Cytokine Staining Starter Kit Mouse Intracellular Cytokine Staining Starter Kit FITC BrdU Flow Kit APC BrdU Flow Kit Description Clone Format Size Cat. No. Related Flow Cytometric Reagents and Buffers anti-brdu 3D4 Purified 0.1 mg anti-brdu FITC Set 100 tests anti-brdu PE Set 100 tests BD Cytofix/Cytoperm Buffer 125 mls BD Perm/Wash Buffer (10 ) 100 mls BD GolgiStop (containing monensin) 0.7 ml BD GolgiPlug (containing brefeldin A) 1.0 ml BD Cytofix Buffer 100 mls BD Pharmingen Stain Buffer (FBS) 500 mls BD Pharmingen Stain Buffer (BSA) 500 mls BrdU Solution 25.0 mg AAD Staining Solution 2.0 ml Propidium Iodide Staining Solution 2.0 ml Related BrdU Products BD Pharmingen BrdU In-Situ Detection Kit 50 tests BD Pharmingen BrdU In-Situ Detection Kit II 200 tests

36 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: Dean, 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. Knüchel and F. Hofstädter 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: 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:

37 20. 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. 37

38 Note 38

39 Note 39

40 R

BrdU Flow Kit

BrdU FLOW KIT Kit Manual FITC -anti-brdu Antibody (Kit Component. No. 23614L) Cytofix/Cytoperm Buffer (Kit Component. No. 2090KE) Perm/Wash Buffer (10x (Kit Component. No. 2091KE) Cytoperm Plus Buffer