Scatter profile of peripheral blood cells from human whole blood, pretreated with RBC Lysis Buffer as recommended.
Note: It is recommended to dilute the RBC Lysis Buffer (10X) just prior to use.
1. Dilute the RBC Lysis Buffer (10X) to a 1X working concentration by adding 1 part RBC Lysis Buffer (10X) to 9 parts room temperature deionized water. Warm the solution to room temperature.
2. Add 2.0 ml of 1X RBC Lysis Buffer to the prepared sample of whole blood (50-100 µL per tube), gently vortex the sample.
3. Incubate for 10-15 minutes at room temperature protected from light.
4. Centrifuge cells at 500 x g for 5 minutes at room temperature. Carefully aspirate the supernatant without disturbing the cell pellet.
5. Wash the cells once in excess with Flow Cytometry Antibody Dilution Buffer #13616, repeat the centrifugation in step 4.
6. Resuspend the cells in the appropriate volume for analysis.
Note: Above outlines the necessary steps for lysis of human whole blood samples. If working with mouse blood or splenocytes, decrease the incubation time (step 3) to 5 minutes. Further optimization may be required.
The 10X solution is stable for 6 months when stored at 4°C.
RBC Lysis Buffer (10X) is a concentrated ammonium chloride-based buffer used for lysing red blood cells in single cell suspension with little to no effect on the nucleated cells. This buffer contains no fixative reagent so the cells remain viable after red blood cell lysis.