CD4+ T cells were defined as CD3+CD4+; CD8+ T cells were defined as CD3+CD8+; CD8+ naïve cells were defined as CD8+CD45RO-CD62L+; CD4 Tregs were defined as CD3+CD4+CD25med-highCD127low; CD4 conventional T cells (Tcons) were defined as CD3+CD4+CD25low-negCD127med-high; natural killer (NK) cells as CD56+CD3−; and B cells as CD19+. Aliquots of anti-coagulated whole blood (ethylenediaminetetraacetic acid [EDTA]) were incubated with fluorophore-conjugated monoclonal antibodies: anti-CD3 V450 (clone UCHT1, BD Biosciences), anti-CD4 APC-H7 (clone RPA-T4, BD Biosciences), anti-CD8 Pacific-Orange (clone RPA-T8, Biolegend), anti-CD25 PE-Cy7 (clone M-A251, BD Biosciences), anti-CD127 PE-Cy5 (clone eBioRDR5, eBioscience) for T-cell subsets; anti-CD56 PE (clone B159, BD Biosciences), anti-CD3 V450 (clone UCHT1, BD Biosciences) for NK/NKT cells, and anti-CD19 APC (clone HIB19, BD Biosciences) for B cells. RBC lysis with BD Pharm Lyse was performed either prior to or following incubation with conjugated antibodies. Flow cytometry analysis utilized FACSCanto II (BD Bioscience) or the Fortessa (BD Bioscience) and FACSDiva software (BD Bioscience). There was a change in the use of flow cytometry machines over the course of the study. Both flow cytometers were validated and results were comparable.
Anti cd25 pe cy7 clone m a251
Manufactured by BD
Anti-CD25 PE-Cy7 (clone M-A251) is a mouse monoclonal antibody conjugated to the fluorochrome Phycoerythrin-Cyanine7 (PE-Cy7). The antibody binds to the CD25 molecule, which is expressed on activated T cells, regulatory T cells, and other immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd3 v450 clone ucht1, by BD (2 mentions)
Anti cd4 apc h7 clone rpa t4, by BD (2 mentions)
Anti cd8 pacific orange clone rpa t8, by BioLegend (2 mentions)
Anti cd127 pe cy5 clone ebiordr5, by Thermo Fisher Scientific (2 mentions)
Anti cd56 pe clone b159, by BD (2 mentions)
Anti cd19 apc clone hib19, by BD (2 mentions)
Fortessa, by BD (1 mentions)
Alexa flour 488, by BD (1 mentions)
3 protocols using anti cd25 pe cy7 clone m a251
1
Comprehensive Immune Cell Profiling
2
Comprehensive Immune Cell Profiling
3
Identifying Regulatory T cells by Flow Cytometry
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Freshly isolated 100 µL of 1 × 106/mL PBMC was stained with 5 or 10 µL of monoclonal antibodies (according to manufacturer's instructions, BD-Pharmingen): anti-CD25 PE-Cy7, clone M-A251; anti-CD4 PE-Cy5; anti-CD127 PE. The samples were incubated for 20 minutes in the dark at room temperature. Next, the cells were washed twice in a washing buffer (PBS supplemented with 0.5% inactivated FBS) for 5 minutes, 250 g. FoxP3 intracellular staining was performed according to manufacturer's instructions (BD-Pharmingen). Briefly, cells were incubated 10 minutes in room temperature in the dark with 2 mL of fixation buffer and then washed in washing buffer, centrifuged at 500 g for 5 minutes, and incubated for 30 minutes in 500 µL of permeabilization buffer in room temperature in the dark. Subsequently, the cells were stained with 20 µL anti-FoxP3 monoclonal antibody (Alexa Flour 488, BD-Pharmingen) for 30 minutes in room temperature in the dark and washed twice before the analysis.
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