BD PharmingenTM DRAQ5TM

Description

Panel 1. Cell cycle analysis of stimulated mouse splenocytes. Mouse splenic leucocytes SPL were cultured 3 days without Left Plot or with Right Plot Purified NA/LE Hamster Anti-Mouse CD3e Cat. No. 553057 and Purified NA/LE Hamster Anti-Mouse CD28 Cat. No. 553294 antibodies. Cells were harvested, stained with Fixable Viability Stain 450 Cat. No. 562247 and stained for BrdU and DNA content with DRAQ5 according to the FITC BrdU Flow Kit Manual Cat. No. 557891/559619 . Viable cells based on Fixable Viability Stain 450 staining were analyzed for BrdU staining and DNA content. Stimulated cells show a large increase in S-phase BrdU+, DRAQ5 2N-4N and G2/M-phase BrdU-, DRAQ5 4N cells. Flow cytometric analysis was performed using a BD LSRFortessa Cell Analyzer System. Panel 2. Flow cytometric analysis of HeLa cell DNA content. HeLa cells in log phase growth were harvested using Gibco Cell Dissociation Buffer Life Technologies and resuspended in complete medium containing 20 muM DRAQ5 15 min, 37C . Cells were pelleted by centrifugation, supernatant was aspirated, and cells were resuspended in DPBS for DNA content analysis using a BD LSRFortessa Cell Analyzer System. Histograms were deconvoluted by FlowJo software into G0/G1, S, and G2/M peaks. Panel 3. Immunofluorescence analysis of E-cadherin expression in human MCF7 cells. Cultured cells from the MCF-7 Breast adenocarcinoma, ATCC HTB-22 cell line were fixed with BD Cytofix Fixation Buffer Cat. No. 554655 , permeabilized with ice-cold BD Phosflow Perm Buffer III Cat. No. 558050 , washed and stained with Purified Mouse Anti-E-Cadherin antibody Cat. No. 610181/610182 at 5 mug/mL. After washing, cells were stained with the second step reagent, BD Horizon BV421 Goat Anti-Mouse Ig Cat. No. 563846 at 1.5 mug/mL pseudo-colored green and DRAQ5 pseudo-colored red as a nuclear counterstain. The image was captured on a Zeiss LSM 710 Confocal Microscope and merged using ImageJ software. Panel 1. Cell cycle analysis of stimulated mouse splenocytes. Mouse splenic leucocytes SPL were cultured 3 days without Left Plot or with Right Plot Purified NA/LE Hamster Anti-Mouse CD3e Cat. No. 553057 and Purified NA/LE Hamster Anti-Mouse CD28 Cat. No. 553294 antibodies. Cells were harvested, stained with Fixable Viability Stain 450 Cat. No. 562247 and stained for BrdU and DNA content with DRAQ5 according to the FITC BrdU Flow Kit Manual Cat. No. 557891/559619 . Viable cells based on Fixable Viability Stain 450 staining were analyzed for BrdU staining and DNA content. Stimulated cells show a large increase in S-phase BrdU+, DRAQ5 2N-4N and G2/M-phase BrdU-, DRAQ5 4N cells. Flow cytometric analysis was performed using a BD LSRFortessa Cell Analyzer System. Panel 2. Flow cytometric analysis of HeLa cell DNA content. HeLa cells in log phase growth were harvested using Gibco Cell Dissociation Buffer Life Technologies and resuspended in complete medium containing 20 muM DRAQ5 15 min, 37C . Cells were pelleted by centrifugation, supernatant was aspirated, and cells were resuspended in DPBS for DNA content analysis using a BD LSRFortessa Cell Analyzer System. Histograms were deconvoluted by FlowJo software into G0/G1, S, and G2/M peaks. Panel 3. Immunofluorescence analysis of E-cadherin expression in human MCF7 cells. Cultured cells from the MCF-7 Breast adenocarcinoma, ATCC HTB-22 cell line were fixed with BD Cytofix Fixation Buffer Cat. No. 554655 , permeabilized with ice-cold BD Phosflow Perm Buffer III Cat. No. 558050 , washed and stained with Purified Mouse Anti-E-Cadherin antibody Cat. No. 610181/610182 at 5 mug/mL. After washing, cells were stained with the second step reagent, BD Horizon BV421 Goat Anti-Mouse Ig Cat. No. 563846 at 1.5 mug/mL pseudo-colored green and DRAQ5 pseudo-colored red as a nuclear counterstain. The image was captured on a Zeiss LSM 710 Confocal Microscope and merged using ImageJ software. Show More Panel 1. Cell cycle analysis of stimulated mouse splenocytes. Mouse splenic leucocytes SPL were cultured 3 days without Left Plot or with Right Plot Purified NA/LE Hamster Anti-Mouse CD3e Cat. No. 553057 and Purified NA/LE Hamster Anti-Mouse CD28 Cat. No. 553294 antibodies. Cells were harvested, stained with Fixable Viability Stain 450 Cat. No. 562247 and stained for BrdU and DNA content with DRAQ5 according to the FITC BrdU Flow Kit Manual Cat. No. 557891/559619 . Viable cells based on Fixable Viability Stain 450 staining were analyzed for BrdU staining and DNA content. Stimulated cells show a large increase in S-phase BrdU+, DRAQ5 2N-4N and G2/M-phase BrdU-, DRAQ5 4N cells. Flow cytometric analysis was performed using a BD LSRFortessa Cell Analyzer System. Panel 2. Flow cytometric analysis of HeLa cell DNA content. HeLa cells in log phase growth were harvested using Gibco Cell Dissociation Buffer Life Technologies and resuspended in complete medium containing 20 muM DRAQ5 15 min, 37C . Cells were pelleted by centrifugation, supernatant was aspirated, and cells were resuspended in DPBS for DNA content analysis using a BD LSRFortessa Cell Analyzer System. Histograms were deconvoluted by FlowJo software into G0/G1, S, and G2/M peaks. Panel 3. Immunofluorescence analysis of E-cadherin expression in human MCF7 cells. Cultured cells from the MCF-7 Breast adenocarcinoma, ATCC HTB-22 cell line were fixed with BD Cytofix Fixation Buffer Cat. No. 554655 , permeabilized with ice-cold BD Phosflow Perm Buffer III Cat. No. 558050 , washed and stained with Purified Mouse Anti-E-Cadherin antibody Cat. No. 610181/610182 at 5 mug/mL. After washing, cells were stained with the second step reagent, BD Horizon BV421 Goat Anti-Mouse Ig Cat. No. 563846 at 1.5 mug/mL pseudo-colored green and DRAQ5 pseudo-colored red as a nuclear counterstain. The image was captured on a Zeiss LSM 710 Confocal Microscope and merged using ImageJ software.