Anti flag fitc antibody

Cells were washed and resuspended in serum free and azide free PBS (Life Technologies) and were stained with A_2AR-FITC, CD4-FITC or CD25-PE for 30 minutes at room temperature. Cells transduced with lentiCRISPR loxP A_2AR virus were marked using anti-FLAG-FITC antibody (Sigma-Aldrich, St. Louis, MO, USA) after fixing and permeabilization. An isotype control was used for each fluorophore used. For FoxP3 staining, cells were fixed and permeabilized for 30 minutes at room temperature using the eBioscience FoxP3 staining buffer set (Affymetrix, San Diego, CA, USA), followed by staining with anti-human FoxP3-APC (Affymetrix). Samples were run on a benchtop BD FACScanto flow cytometer (BD Biosciences, San Diego, CA, USA) and a FACScan (BD Biosciences). Analysis was performed using FlowJo FACS analysis software (FlowJo LLC, Ashland, OR, USA) and CellQuestPro (BD Biosciences).

Cell surface expression for each mutant was monitored using flow cytometry. The expressed Hi5 cells (10 μL) were incubated with 10 μL anti-FLAG-FITC antibody (Sigma), which is diluted with PBS containing 4% BSA at a final ratio of 1:1000, at 4 °C for 15 min, and 180 μL 1× PBS was then added to the cells. The surface expression of each mutant was monitored by detecting the fluorescent intensity of FITC using a BD ACCURI C6.