1. Each sample is represented in two tubes: one stained with the isotype controls and the other stained with specific markers.
2. The appropriate volume of adjusted cells (equivalent to 1 X 106 cells) is pipetted into each of the two 12 X 75-mm flow tubes.
3. The control tube is incubated with 5 ^L of IgG1 FITC, IgG1 PerCP, and IgG1 APC. The markers tube is incubated with 10 ^L of CD19 FITC, CD20-PE, and CD3-APC. Incubations take place for 15 min at room temperature in the dark.
4. The samples are then incubated with 2 mL lysing reagent for 10 min at room temperature in the dark.
5. The samples are washed and resuspended in 500 flow PBS for analysis.
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