Fitc cd19 6d5

Eighty microliters of blood were collected by tail vein incision in a tube containing 20 μl of 3.8% sodium citrate. Erythrocytes were lysed by adding 600 μl of erythrocyte lysis solution (Qiagen, Valencia, CA) for 5 min in agitation. Then blood was centrifuged at 260 g for 5 min and the pellet was resuspended in 10 ml of cold PBS. Cells were centrifuged at 260 g for 5 min and then resuspended in 100 μl of FACS buffer (1% FBS in PBS) containing the appropriate antibodies. Cells were stained at 4°C in the dark for 1 h and then pelleted after adding 1 ml of FACS buffer. Cells were resuspended in 500 μl of FACS buffer and analyzed at BD FACSCalibur flow cytometer. B-lymphocytes were stained with APC Cd45 (30-F11, #103112, 1:100; BioLegend), FITC Cd19 (6D5, #115506, 1:100; BioLegend), and PE Cd39 (5F2, #12-3390-80, 1:100; eBioscience). APC rat IgG2b (RTK4530, #400611, 1:100; BioLegend), FITC rat IgG2a (R35-95, #554688, 1:100; BD Biosciences), and PE mouse IgG1 (P3.6.2.8.1, #12-4714-41, 1:100; eBioscience) were used as isotype controls. Thirty thousand events from lymphocyte gate were acquired in each analysis.