Cd8 apc cy7 clone sk1

Cryopreserved PBMCs were thawed and 1x10⁶ cells per sample washed with PBS containing 2% FCS, 2 mM EDTA, and 0.01% sodium azide at room temperature. After Fc receptor blocking with Gamunex (human immunoglobulin; GRIFOLS) and labeling of dead cells with ethidium monoazide (EMA; Biotinum), the cells were stained with the following titrated monoclonal antibodies: CD3-A700 (clone UCHT1, Biolegend), CD56-FITC (clone HCD56, Biolegend), CD4-PerCP (clone SK3, BD Bioscience), CD8-APC-Cy7 (clone SK1, Biolegend) and PD-1-BV711 (clone EH12.2H7, Biolegend). Lastly, after additional washing the samples were measured with a LSRII cytometer (BD) and processed with BD FACSDiva software 6.1.3. Single color controls were used for automatically calculated compensation.

The following antibodies were used for phenotyping and proliferation assays: anti-CD3-PE/Cy7 (clone SK7, BioLegend, San Diego, CA), anti-CD4-PerCP (clone SK3, BD Pharmigen, San Jose, CA), anti-CD4 A700 (clone RPA T4, BioLegend), anti-CD19 APC (clone HIB19, BD Pharmigen), anti-CD25 APC (clone 2A3, BD Pharmigen), anti-CD71 FITC (clone CY1G4, BioLegend), anti-Myc FITC or APC (clone 9B11, Cell Signaling, Danvers, MA), anti-FMC19 idiotype APC (Juno Therapeutics), anti-EGFRt PE (Juno Therapeutics), anti-CD28 APC (clone 28.2, Biolegend), and CD8 APC-Cy7 (clone SK1, BioLegend). DAPI (ThermoFisher, Waltham, MA) was used to stain dead cells for exclusion during analysis. Flow cytometric analyses were performed on an LSR II Flow Cytometer System (BD Biosciences). Fluorescence-activated cell sorting was performed on an FACSAria III Cell Sorter (BD Biosciences). All flow cytometry data were analyzed using the FlowJo software (Tree Star, Ashland, OR).