BD PharmingenTM Apoptosis, DNA Damage and Cell Proliferation Kit

Description

Figure 1. Overview: The BD Pharmingen Apoptosis, DNA Damage, and Cell Proliferation Kit Staining Protocol. 1 Recipe for Freezing Media: 10 % dimethyl sulfoxide DMSO + 90 % heat-inactivated Fetal Bovine Serum FBS . 2 The immunofluorescent staining of cell surface antigens can be done at the same time as staining intracellular antigens provided that the antibodies recognize paraformaldehyde-fixed epitopes. 3 If staining for total DNA content is not desired, then the DAPI staining step can be omitted and fluorescent data for another parameter can then be measured in the UV or Violet channel. Figure 2. Multiparameter cell cycle analysis of stimulated human peripheral blood mononuclear cells PBMC . PBMC were stimulated with Purified NA/LE Mouse Anti-Human CD3 Cat. No. 555329 and Purified NA/LE Mouse Anti-Human CD28 Cat. No. 555725 antibodies for 3 days. The activated cells were harvested and re-plated in complete medium and labeled with 50 mu M BrdU for 1 hr. The cells were then harvested and analyzed by immunofluorescent staining and multicolor flow cytometric analysis using a BD LSR II Flow Cytometer System. Panel A: DAPI versus BrdU PerCP-Cy5.5 staining profile for activated PBMC. Panel B: BrdU PerCP-Cy5.5 versus H2AX pS139 Alexa Fluor 647 profile. Panel C: Cleaved PARP Asp214 PE versus BrdU PerCP-Cy5.5 profile. Panel D: Cleaved PARP Asp214 PE versus H2AX pS139 Alexa Fluor 647 profile. BrdU-positive cells are color-gated red whereas BrdU-negative cells are colored blue. Figure 1. Overview: The BD Pharmingen Apoptosis, DNA Damage, and Cell Proliferation Kit Staining Protocol. 1 Recipe for Freezing Media: 10 % dimethyl sulfoxide DMSO + 90 % heat-inactivated Fetal Bovine Serum FBS . 2 The immunofluorescent staining of cell surface antigens can be done at the same time as staining intracellular antigens provided that the antibodies recognize paraformaldehyde-fixed epitopes. 3 If staining for total DNA content is not desired, then the DAPI staining step can be omitted and fluorescent data for another parameter can then be measured in the UV or Violet channel. Show More Figure 2. Multiparameter cell cycle analysis of stimulated human peripheral blood mononuclear cells PBMC . PBMC were stimulated with Purified NA/LE Mouse Anti-Human CD3 Cat. No. 555329 and Purified NA/LE Mouse Anti-Human CD28 Cat. No. 555725 antibodies for 3 days. The activated cells were harvested and re-plated in complete medium and labeled with 50 mu M BrdU for 1 hr. The cells were then harvested and analyzed by immunofluorescent staining and multicolor flow cytometric analysis using a BD LSR II Flow Cytometer System. Panel A: DAPI versus BrdU PerCP-Cy5.5 staining profile for activated PBMC. Panel B: BrdU PerCP-Cy5.5 versus H2AX pS139 Alexa Fluor 647 profile. Panel C: Cleaved PARP Asp214 PE versus BrdU PerCP-Cy5.5 profile. Panel D: Cleaved PARP Asp214 PE versus H2AX pS139 Alexa Fluor 647 profile. BrdU-positive cells are color-gated red whereas BrdU-negative cells are colored blue. Show More Figure 1. Overview: The BD Pharmingen Apoptosis, DNA Damage, and Cell Proliferation Kit Staining Protocol. 1 Recipe for Freezing Media: 10 % dimethyl sulfoxide DMSO + 90 % heat-inactivated Fetal Bovine Serum FBS . 2 The immunofluorescent staining of cell surface antigens can be done at the same time as staining intracellular antigens provided that the antibodies recognize paraformaldehyde-fixed epitopes. 3 If staining for total DNA content is not desired, then the DAPI staining step can be omitted and fluorescent data for another parameter can then be measured in the UV or Violet channel. Figure 2. Multiparameter cell cycle analysis of stimulated human peripheral blood mononuclear cells PBMC . PBMC were stimulated with Purified NA/LE Mouse Anti-Human CD3 Cat. No. 555329 and Purified NA/LE Mouse Anti-Human CD28 Cat. No. 555725 antibodies for 3 days. The activated cells were harvested and re-plated in complete medium and labeled with 50 mu M BrdU for 1 hr. The cells were then harvested and analyzed by immunofluorescent staining and multicolor flow cytometric analysis using a BD LSR II Flow Cytometer System. Panel A: DAPI versus BrdU PerCP-Cy5.5 staining profile for activated PBMC. Panel B: BrdU PerCP-Cy5.5 versus H2AX pS139 Alexa Fluor 647 profile. Panel C: Cleaved PARP Asp214 PE versus BrdU PerCP-Cy5.5 profile. Panel D: Cleaved PARP Asp214 PE versus H2AX pS139 Alexa Fluor 647 profile. BrdU-positive cells are color-gated red whereas BrdU-negative cells are colored blue.