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Comments:
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Each sample was first blocked with 100ul of 5% Human TruStain FcX (Biolegend) in wash buffer (4% FCS in dPBS) for 7min at room temperature. Samples were pelleted (200g, 5 min, 4°C) and resuspended in 100ul of the following binding buffer: 20ul (1 test volume) mouse anti-human CD19-PE (BD Biosciences), 20ul (1 test volume) mouse anti-human CD4-FITC (BD Biosciences), 5ul (1 test volume) 7-AAD (BD Biosciences), and 45ul wash buffer. Samples were incubated in the dark at 4°C for 20 min. Following incubation, samples were washed twice with wash buffer before sorting with BD FACSAriaII at a standard flow rate for PE, FITC, and PerCP-Cy5.5-A channels. Standard FSC and SSC gates were used to identify lymphocytes. CD4+/CD19-/7-AAD- cells and CD19+/CD4-/7-AAD- cells were collected for subsequent analysis.
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