Anti cd3 apc cy7

Agonist or inhibitor-treated cells were incubated with undiluted goat serum for 30 mins at 4°C, then washed in FACS buffer (1% BSA in PBS). Cells were resuspended with FACS buffer containing anti-CCR1-PE, anti-CD3-APC-Cy7, or anti-CD14-Pacific Blue (BD Biosciences Pharmingen, Palo Alto, CA), or anti-CCR2-PE antibody (R&D systems, Minneapolis, MN), and incubated at 4°C for 30 mins. After washing twice with FACS buffer, the cells were resuspended in FACS buffer, and analyzed using FACS Calibur, FACS Aria, or LSRII flow cytometers (BD Biosciences). A minimun of 10,000 gated cells were analyzed per sample for the relevant cell surface receptor expression. For quantification analysis of CCR1 and CCR2, the mean fluorescent intensities of CCR1-PE or CCR2-PE labeled cells were converted to the number of antibody bound receptors with Quantibrite PE calibration beads (Becton Dickinson, San Jose, CA) according to the manufacturer’s instructions.

Clone cells were washed and left in fresh RPMI 1640 CM (5% AB serum) without IL-2 to rest for 5 hours or overnight before being stimulated with RL9 peptide pulsed K562-E (50μM, 20-24 hours at 27°C), K562-E-RL9 or K562-D-RL9 cells at a clone: K562 cell ratio of 1:3 for 1 hour, followed by addition of 5μg/ml Brefeldin A (Biolegend) and 5μg/ml GolgiStop (BD Biosciences) for an additional 8 hours at 37°C. For CD107 staining, anti-CD107a-BV421 and anti-CD107b-BV421 (Biolegend) antibodies were added at the beginning of the co-culture. After 9 hours incubation, cells were washed with PBS and stained with Live/Dead Fixable Aqua, anti-CD8-PerCP-Cy5.5 and anti-CD3-APC-Cy7 for 30 min at RT first, then fixed/permeabilized with Cytofix/Cytoperm 1x Solution (BD Biosciences) for 10 min at 4°C, and stained in Permwash 1x Solution (BD Biosciences) with anti-TNFα-PE, anti-IFN-γ–FITC and anti-CD137-BV650 (All BioLegend) for 30 min at RT. After being washed with PBS and fixed with 2% paraformaldehyde, samples were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software v10.3 (Tree Star). In the selected assays to determine HLA-E restriction, K562-E cells were pre-incubated with the VL9 canonical signal peptide (VMAPRTLVL, 50μM, 3 hours at 27°C) prior to addition of RL9 peptide.