BD PhosflowTM PE Mouse anti-CD22 (pY822)

Description

Analysis of CD22 pY822 in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells PBMC were either stimulated right panel by cross-linking of surface IgM with purified Goat F ab 2 Anti-Human IgM SouthernBiotech at 37C for 2 minutes or unstimulated left panel . The cells were fixed with pre-warmed BD Phosflow Lyse/Fix Buffer Cat. No. 558049 for 10 minutes at 37oC, permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with PE Mouse anti-CD22 pY822, Cat. No. 560076 and Alexa Fluor 647 Mouse anti-human CD20 Cat. No. 558054 . For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation is restricted to the stimulated CD20-positive B lymphocytes. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. We have also demonstrated that this antibody conjugate stains mouse CD22 pY837 in splenocytes that are activated by IgM cross-linking data not shown . The specificity of mAb 12a/CD22 was confirmed by western blot analysis using unconjugated antibody at 0.5 mug/ml on lysates from control lane 1 and anti-IgM-stimulated lane 2 mouse splenocytes. Mouse CD22 pY837 is identified as a band of 140 kDa in the activated splenocytes. Cross-reactivity to human CD22 pY822 was demonstrated by western blot analysis on lysates from unstimulated and pervanadate-stimulated Daudi Burkitts lymphoma cells data not shown . Analysis of CD22 pY822 in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells PBMC were either stimulated right panel by cross-linking of surface IgM with purified Goat F ab 2 Anti-Human IgM SouthernBiotech at 37C for 2 minutes or unstimulated left panel . The cells were fixed with pre-warmed BD Phosflow Lyse/Fix Buffer Cat. No. 558049 for 10 minutes at 37oC, permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with PE Mouse anti-CD22 pY822, Cat. No. 560076 and Alexa Fluor 647 Mouse anti-human CD20 Cat. No. 558054 . For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation is restricted to the stimulated CD20-positive B lymphocytes. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. We have also demonstrated that this antibody conjugate stains mouse CD22 pY837 in splenocytes that are activated by IgM cross-linking data not shown . Show More The specificity of mAb 12a/CD22 was confirmed by western blot analysis using unconjugated antibody at 0.5 mug/ml on lysates from control lane 1 and anti-IgM-stimulated lane 2 mouse splenocytes. Mouse CD22 pY837 is identified as a band of 140 kDa in the activated splenocytes. Cross-reactivity to human CD22 pY822 was demonstrated by western blot analysis on lysates from unstimulated and pervanadate-stimulated Daudi Burkitts lymphoma cells data not shown . Show More Analysis of CD22 pY822 in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells PBMC were either stimulated right panel by cross-linking of surface IgM with purified Goat F ab 2 Anti-Human IgM SouthernBiotech at 37C for 2 minutes or unstimulated left panel . The cells were fixed with pre-warmed BD Phosflow Lyse/Fix Buffer Cat. No. 558049 for 10 minutes at 37oC, permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with PE Mouse anti-CD22 pY822, Cat. No. 560076 and Alexa Fluor 647 Mouse anti-human CD20 Cat. No. 558054 . For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation is restricted to the stimulated CD20-positive B lymphocytes. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. We have also demonstrated that this antibody conjugate stains mouse CD22 pY837 in splenocytes that are activated by IgM cross-linking data not shown . The specificity of mAb 12a/CD22 was confirmed by western blot analysis using unconjugated antibody at 0.5 mug/ml on lysates from control lane 1 and anti-IgM-stimulated lane 2 mouse splenocytes. Mouse CD22 pY837 is identified as a band of 140 kDa in the activated splenocytes. Cross-reactivity to human CD22 pY822 was demonstrated by western blot analysis on lysates from unstimulated and pervanadate-stimulated Daudi Burkitts lymphoma cells data not shown .