BD PhosflowTM Alexa Fluor 647 Mouse anti-S6 (pS240)

Description

Analysis of S6 pS240 in activated human peripheral blood mononuclear cells PBMC . PBMC were isolated by density gradient centrifugation Ficoll-Paque PLUS, Cat. No. 17-1440-02 and either left untreated open histogram or treated with PMA Sigma-Aldrich, Cat. No. P8139 at 50 nM/106 cells for 30 minutes shaded histogram . Cells were then fixed in BD Cytofix buffer Cat. No. 554655 at 37C for 10 minutes, then permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with Alexa Fluor 647 Mouse anti-S6 pS240, Cat. No. 560432 . For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. Immunofluorescent staining of human cell line. HeLa cells ATCC CCL-2 were seeded in a 96-well imaging plate Cat. No. 353219 at 10,000 cells per well. After overnight serum starvation, some wells were cultured for 30 minutes with 20 % fetal bovine serum FBS, top panel and others were not lower panel . The cells were fixed with BD Cytofix buffer Cat. No. 554655 , permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 , stained with Alexa Fluor 647 Mouse anti-S6 pS240 psedocolored magenta , and the nuclei were counterstained with Hoechst 33342 pseudo colored blue . The images were captured on a BD Pathway 435 High-Content Bioimager System with a 20x objective and merged using BD Attovision software. The staining worked with the cold methanol and the Triton X-100 Perm/Wash protocols. Western blot analysis of S6 pS240 . The specificity of mAb N4-41 was confirmed by western blot analysis using unconjugated Mouse anti-S6 pS240 antibody on lysates from untreated lane 1 or PMA-treated lane 2 PBMC. S6 pS240 is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells. Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656 or 612657 was the gel-loading control. Analysis of S6 pS240 in activated human peripheral blood mononuclear cells PBMC . PBMC were isolated by density gradient centrifugation Ficoll-Paque PLUS, Cat. No. 17-1440-02 and either left untreated open histogram or treated with PMA Sigma-Aldrich, Cat. No. P8139 at 50 nM/106 cells for 30 minutes shaded histogram . Cells were then fixed in BD Cytofix buffer Cat. No. 554655 at 37C for 10 minutes, then permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with Alexa Fluor 647 Mouse anti-S6 pS240, Cat. No. 560432 . For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. Show More Immunofluorescent staining of human cell line. HeLa cells ATCC CCL-2 were seeded in a 96-well imaging plate Cat. No. 353219 at 10,000 cells per well. After overnight serum starvation, some wells were cultured for 30 minutes with 20 % fetal bovine serum FBS, top panel and others were not lower panel . The cells were fixed with BD Cytofix buffer Cat. No. 554655 , permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 , stained with Alexa Fluor 647 Mouse anti-S6 pS240 psedocolored magenta , and the nuclei were counterstained with Hoechst 33342 pseudo colored blue . The images were captured on a BD Pathway 435 High-Content Bioimager System with a 20x objective and merged using BD Attovision software. The staining worked with the cold methanol and the Triton X-100 Perm/Wash protocols. Show More Show More Western blot analysis of S6 pS240 . The specificity of mAb N4-41 was confirmed by western blot analysis using unconjugated Mouse anti-S6 pS240 antibody on lysates from untreated lane 1 or PMA-treated lane 2 PBMC. S6 pS240 is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells. Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656 or 612657 was the gel-loading control. Show More Analysis of S6 pS240 in activated human peripheral blood mononuclear cells PBMC . PBMC were isolated by density gradient centrifugation Ficoll-Paque PLUS, Cat. No. 17-1440-02 and either left untreated open histogram or treated with PMA Sigma-Aldrich, Cat. No. P8139 at 50 nM/106 cells for 30 minutes shaded histogram . Cells were then fixed in BD Cytofix buffer Cat. No. 554655 at 37C for 10 minutes, then permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with Alexa Fluor 647 Mouse anti-S6 pS240, Cat. No. 560432 . For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. Immunofluorescent staining of human cell line. HeLa cells ATCC CCL-2 were seeded in a 96-well imaging plate Cat. No. 353219 at 10,000 cells per well. After overnight serum starvation, some wells were cultured for 30 minutes with 20 % fetal bovine serum FBS, top panel and others were not lower panel . The cells were fixed with BD Cytofix buffer Cat. No. 554655 , permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 , stained with Alexa Fluor 647 Mouse anti-S6 pS240 psedocolored magenta , and the nuclei were counterstained with Hoechst 33342 pseudo colored blue . The images were captured on a BD Pathway 435 High-Content Bioimager System with a 20x objective and merged using BD Attovision software. The staining worked with the cold methanol and the Triton X-100 Perm/Wash protocols. Western blot analysis of S6 pS240 . The specificity of mAb N4-41 was confirmed by western blot analysis using unconjugated Mouse anti-S6 pS240 antibody on lysates from untreated lane 1 or PMA-treated lane 2 PBMC. S6 pS240 is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells. Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656 or 612657 was the gel-loading control.