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Next, cells were centrifuged and washed twice with FACS buffer (1x PBS, 0.05% Bovine Serum Albumin, 0.05% NaN 3 ), resuspended in FACS buffer, and analyzed using either a FACSCalibur machine (BD Biosciences) or a CytoFlex machine (Beckman-Coulter Life Sciences) and the FCS Express software (De Novo Software)