Cd69 apc

Cell surface receptor expression was measured with CD69 (APC), or CD45 (PE) antibodies (BD Biosciences) using the manufacturer’s recommendations. Cells were incubated on ice for 1 hour, washed 3 times with PBS and fixed in 2% paraformaldehyde (Polysciences). Purified extracellular cellular vesicles (EV) were stained with either anti-CD63 exo-flow staining kit (Systems Biosciences) or CFSE dye (5μM) for 15 minutes at 37°C. EVs were washed in PBS four times and concentrated using Amicon 100K filter units (Millipore). Data was acquired on BD LSR II flow cytometer using single stained CompBeads (BD Biosciences) for compensation. At least 10,000 total events were collected in each experiment and the FlowJo software program (Tree Star Inc.) was used for data analysis. All flow cytometry experiments were repeated at least three times with consistent results.

The cultures were collected, washed, incubated for 15 min with mouse mAbs against human CD3-PerCP, CD56-FITC, or PE, CD69-APC, CD16-PE (BD Biosciences, USA), and NKG2D-PE (BioLegend, USA). NK cells were incubated with CD158a-PE and CD158b-PE (BD Pharmingen, USA), CIK cells were incubated with CD4-PE and CD8-APC (BD Biosciences) and γδ T cells were incubated with Vγ9-FITC (BD Pharmingen), CD4-PE, and CD8-APC. Isotype-matched antibodies were used as controls. Perforin and granzyme B detection was performed according to the BD Cytofix/Cytoperm™ Kit manual (BD Biosciences). Briefly, NK, CIK, and γδ T cells were harvested and adjusted to 1 × 10⁶ cells/mL in RPMI-1640 medium containing 10 % fetal calf serum, and incubated 0.1 % GolgiStop (BD Biosciences) for 4 h. After pre-incubation with 10 % normal human serum, cells were stained with mAbs to identify NK (CD3⁻CD56⁺), CIK (CD3⁺CD56⁺), and γδ T cells (CD3⁺Vγ9⁺), followed by intracellular staining for perforin-PE and granzyme B-PE (BD Pharmingen), and the corresponding isotype antibodies to determine intracellular cytokine levels.
Flow cytometry data acquisition was performed on a BD FACS Calibur (BD Biosciences) with Cell Quest Pro software. Analysis was performed with FlowJo software (Tree Star, USA).

On day 0, 7, and 14, the PBMCs from the T‐cell stimulation assay were analyzed in flow‐cytometry by anti‐CD3‐FITC, CD8‐PerCP, CD45RO‐PE, CD45RA‐APC‐H7, and CD69‐APC antibodies (BD, Heidelberg, Germany) with a FACS Canto II.
Cytotoxicity of effector cells that were stimulated for 14 days was tested by measuring LDH release with the CytoTox 96 Non‐Radioactive Cytotoxicity Assay (Promega, Madison, Wisconsin) following incubation with peptide loaded T2/mHLA‐A*24:02 cells at a ratio of 5:1. The cells were incubated for 4 hours at 37°C and LDH release was measured according to kit instructions.