BD PharmingenTM Human Regulatory T Cell Sorting Kit

Description

Example Sort Data. Example data was derived from PBMC isolated from sodium heparin anticoagulated whole blood. Cell viability was > 94 % and cells were stained at 100 million cells/mL with the BD Human Regulatory T cell Sorting Kit. The stained cells were sorted on a FACSAria flow cytometer using FACSDiva software. Presort: Lymphocytes were gated for expected morphology using a FSC-A vs. SSC-A Lymphocyte Gate followed by doublet discrimination using FSC-W and SSC-W parameters. Next, CD4+ lymphocytes were gated followed by gating of the CD25highCD127low T regulatory Treg cells.This population of CD4+CD25highCD127low can be directly sorted as shown. Alternately, CD45RA- and CD45RA+ staining patterns can provide an additional interrogation parameter used to identify CD4+CD25highCD127lowCD45RA+ and CD4+CD25highCD127lowCD45RA- populations as shown . Cells were sorted in the purity mode using a 70 micron nozzle with a rate of 8,000-to-11,000 events per second. Post Sort: Each sorted fraction was counted, divided into two fractions, and washed and suspended in culture media or wash buffer for cell culture or analysis respectively. At least 30,000 cells were suspended in 300 muL of wash buffer for purity measurements. Slight adjustments of gates were required to properly visualize the sorted fractions. The flexibility of the kit allows for many sorting configurations. CD25 expression can vary depending upon donor and the application. An alternative gating strategy isolating CD45RA-CD25high activated T regs is shown in the upper right hand corner. Example Sort Data. Example data was derived from PBMC isolated from sodium heparin anticoagulated whole blood. Cell viability was > 94 % and cells were stained at 100 million cells/mL with the BD Human Regulatory T cell Sorting Kit. The stained cells were sorted on a FACSAria flow cytometer using FACSDiva software. Presort: Lymphocytes were gated for expected morphology using a FSC-A vs. SSC-A Lymphocyte Gate followed by doublet discrimination using FSC-W and SSC-W parameters. Next, CD4+ lymphocytes were gated followed by gating of the CD25highCD127low T regulatory Treg cells.This population of CD4+CD25highCD127low can be directly sorted as shown. Alternately, CD45RA- and CD45RA+ staining patterns can provide an additional interrogation parameter used to identify CD4+CD25highCD127lowCD45RA+ and CD4+CD25highCD127lowCD45RA- populations as shown . Cells were sorted in the purity mode using a 70 micron nozzle with a rate of 8,000-to-11,000 events per second. Post Sort: Each sorted fraction was counted, divided into two fractions, and washed and suspended in culture media or wash buffer for cell culture or analysis respectively. At least 30,000 cells were suspended in 300 muL of wash buffer for purity measurements. Slight adjustments of gates were required to properly visualize the sorted fractions. The flexibility of the kit allows for many sorting configurations. CD25 expression can vary depending upon donor and the application. An alternative gating strategy isolating CD45RA-CD25high activated T regs is shown in the upper right hand corner. Show More Example Sort Data. Example data was derived from PBMC isolated from sodium heparin anticoagulated whole blood. Cell viability was > 94 % and cells were stained at 100 million cells/mL with the BD Human Regulatory T cell Sorting Kit. The stained cells were sorted on a FACSAria flow cytometer using FACSDiva software. Presort: Lymphocytes were gated for expected morphology using a FSC-A vs. SSC-A Lymphocyte Gate followed by doublet discrimination using FSC-W and SSC-W parameters. Next, CD4+ lymphocytes were gated followed by gating of the CD25highCD127low T regulatory Treg cells.This population of CD4+CD25highCD127low can be directly sorted as shown. Alternately, CD45RA- and CD45RA+ staining patterns can provide an additional interrogation parameter used to identify CD4+CD25highCD127lowCD45RA+ and CD4+CD25highCD127lowCD45RA- populations as shown . Cells were sorted in the purity mode using a 70 micron nozzle with a rate of 8,000-to-11,000 events per second. Post Sort: Each sorted fraction was counted, divided into two fractions, and washed and suspended in culture media or wash buffer for cell culture or analysis respectively. At least 30,000 cells were suspended in 300 muL of wash buffer for purity measurements. Slight adjustments of gates were required to properly visualize the sorted fractions. The flexibility of the kit allows for many sorting configurations. CD25 expression can vary depending upon donor and the application. An alternative gating strategy isolating CD45RA-CD25high activated T regs is shown in the upper right hand corner.