Two 100-μl samples of thawed blood were placed in two tubes for staining. They were washed three times with the use of Sorvall cell washers. Then 10 μl of rabbit immunoglobulin was added and incubated at 37°C for 30 minutes, followed by 10 μl of an isotypic cocktail (consisting of fluorescein isothiocyanate–conjugated IgG, phycoerythrin-conjugated IgG, peridinin–chlorophyll–protein cyanine 5.5–conjugated anti-CD19 antibodies, phycoerythrin–cyanine 7–conjugated IgG, allophycocyanin-conjugated IgG, and allophycocyanin H7–conjugated anti-CD45 antibodies), which was added to one tube; the second tube was stained with the custom conjugated six-color antibodies cocktail (consisting of fluorescein isothiocyanate–conjugated anti-CD5, phycoerythrin-conjugated antilambda, peridinin–chlorophyll–protein cyanine 5.5–conjugated anti-CD19 antibodies, phycoerythrin–cyanine 7–conjugated CD10, allophycocyanin-conjugated anti-kappa, and allophycocyanin H7–conjugated anti-CD45 antibodies) and incubated for 20 minutes. All antibodies were obtained from BD Biosciences. After this procedure, 1 ml of redcell lyse buffer was added to each tube and incubated for 10 minutes and then washed with Sorvall cell washer. Cells were then resuspended in 350 μl of phosphate-buffered saline and acquired with the use of FACSCanto flow cytometry; daily quality control and assurance were carried out with the use of seven-color setup beads (BD Biosciences).
