Recommended Protocols for Phospho Protein Detection in Human Cells

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Recommended Protocols for Phospho Protein Detection in Human Cells Depending on subcellular localization of the phospho protein of interest, as well as epitope susceptibility to cell fixing and permeabilizing agents, we recommend different protocols for our antibodies. For phenotyping of cell populations in multi color experiments, please use the table on pages 27 30, showing our human cell surface markers tested under different fixation and permeabilization conditions. Protocol I II III Fix buffers used for PBMCs: BD Phosflow Fix Buffer I BD Cytofix Buffer BD Cytofix Buffer (557870) (554655) (554655) Fix buffers used for Whole Blood: BD Phosflow Lyse/Fix BD Phosflow Lyse/Fix BD Phosflow Lyse/Fix Buffer (558049) Buffer (558049) Buffer (558049) Perm buffers used: BD Phosflow Perm/Wash I BD Phosflow Perm II BD Phosflow Perm III Buffer (557885) Buffer (558052) Buffer (558050) Specificity Clone Fluorochrome ERK 1/2 (pt202/py204) 20a LCK (py505) 4/Lck-Y505 p38 MAPK (pt180/py182) 36 ZAP70 (py319)/syk (py352) 17a Alexa 488 + + + C-Cbl (py700) 47/c-Cbl (Y700) Alexa 488 + + Stat1 (py701) 4a Alexa 488 - +/- + PE +/- + + Stat3 (py705) 4 Alexa 488 - +/- + Stat3 (py727) 49 Alexa 488??? Alexa 647??? Stat4 (py693) 38/p-Stat4 Alexa 488 - - + Alexa 647 + Stat5 (py694) 47 Alexa 488 - +/- + Stat6 (py641) 18 Alexa 488 - +/- + /- 26 www.bdbiosciences.com

Validated Human Cell Surface Markers for Analysis Many cell surface markers are sensitive to fixation/permeabilization procedures and lose recognition by relevant antibodies afterwards. For your convenience, many antibodies conjugated to various fluorochromes have been tested under several conditions. The results are summarized below. Protocol I II III Fix buffers used for PBMCs: BD Phosflow Fix Buffer I BD Cytofix Buffer BD Cytofix Buffer (557870) (554655) (554655) Fix buffers used for Whole Blood: BD Phosflow Lyse/Fix BD Phosflow Lyse/Fix BD Phosflow Lyse/Fix Buffer (558049) Buffer (558049) Buffer (558049) Perm buffers used: BD Phosflow Perm/Wash I BD Phosflow Perm II BD Phosflow Perm III Buffer (557885) Buffer (558052) Buffer (558050) Specificity Clone Fluorochrome CD3 SK7 PerCP + + +/- PE-Cy7 + + +/- APC-Cy7 + CD3 UCHT1 FITC + + + Alexa 700 + CD3 HIT3a FITC - - CD4 L200 FITC + + + PerCP + + + PerCPCy5.5 + + + CD4 RPA-T4 FITC + + + CD4 SK3 PerCP + + + PE-Cy7 + + + CD5 UCHT2 CD8 HIT8a FITC + + + Chapter 1 27

Specificity Clone Fluorochrome I II III CD8 RPA-T8 FITC + + + PE-Cy7 + + - APC-Cy7 + Alexa 700 + CD8 SK1 PerCP + +/- CD11a/LFA-1 G43-25B PE + +/- - CD11a/LFA-1 Hi111 PE + +/- - - - CD11b ICRF44 PE + +/- +/- - - PE-Cy7 + - Alexa 488 + CD11c BLy6 PE + +/- +/- +/- +/- CD13 WM-15 PE +/- +/- CD14 M5E2 FITC + +/- - PE*** + +/- - PerCP-Cy5.5 + PE-Cy7 + - Alexa 488*** + +/- CD14 MoP9 FITC*** - PE*** + - - CD15 W6D3 PE + + CD15 H198 PE + + CD16 3G8 PE + +/- - FITC +/- +/- PE-Cy7 + - - Alexa 488 +/- CD16 B73.1 PE*** + +/- - CD16 NKP15 FITC - - CD18 6.7 CD19* HIB19 PE*** + +/- - FITC +/- +/- PerCP-Cy5.5 +/- PE-Cy7 - Alexa 488*** - Alexa 647*** +/- CD19 SJ25C1 PE*** + +/- - PE-Cy7 +/- - - CD20 2H7 FITC +/- - - PE + - - CD20 L27-28 www.bdbiosciences.com

Specificity Clone Fluorochrome I II III CD20 (I/C) H1(FB1) CD23 M-L233 PE + +/- - CD25 M-A251 FITC + + CD27 L128 FITC + + + CD27 MT271 - CD28 CD28.2 FITC - - CD31 WM59 PE + +/- +/- CD33 WM53 PE + +/- +/- CD33 p67.6 PE +/- +/- CD34 8G12 PE + +/- - CD34 581 CD38 HB7 FITC - - - - CD38 HIT2 - CD40 5C3 FITC - - - - CD44 515 PE + + CD44 G44-26 PE + + + CD45 HI30 CD45RA 5H9 FITC + + CD45RA HI100 FITC + + + CD45RO UCHL1 FITC + + + CD54 HA58 PE +/- +/- +/- CD56 B159 - FITC - PE-Cy7 - - Alexa 488 - Alexa 647 - CD56 MY31 PE +/- +/- CD56 NCAM16.2 - CD69 FN50 FITC - - PE +/- - - - CD79a (I/C) HM47 CD80 L307.4 PE DC+ DC+ Chapter 1 29

Specificity Clone Fluorochrome I II III CD83 HB15e PE + +/- +/- CD86 2331 CD86 IT2.2 CD94 HP-3D9 FITC + + + CD138 Mi15 CD195 2D7 FITC - - - PE-Cy7 +/- - - CD195 3A9 - DC-SIGN DCN46 CCR7 - HLA-DR G46-6 FITC + + +/- PE + + * HLA-DR Tu36 PE + +/- - IgD IA6-2 - IgM G20-127 PE + +/- +/- Legend: DC Dendritic Cells + Positive - Negative +/- Inconclusive Stimulators used for Multi-Color Analysis of Human Whole Blood or PBMCs PMA IFNα IL-2 IL-4 IL-6 CD3/CD28 Activation perk 1/2 pp38 MAPK pstat1 pstat1 (+/-) pstat3 pstat3 pstat5 pstat5 pstat6 pzap70 plck Bulk Conc. Final Conc. For PBMC Final Conc. For Whole Blood PMA 1mg/ml 40nM 400nM IFNα 0.2mg/ml 10,000U/ml 40,000U/ml (3.8 10 8 U/ml) IL-2 0.03µg/µl 0.1µg/ml IL-4 0.1µg/µl 0.1µg/ml IL-6 0.2µg/µl 0.1µg/ml 30 www.bdbiosciences.com

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