10x RBC Lysis/Fixation Solution

Background

10xRBC Lysis/Fixation Solution is designed for FCM sample processing of human whole blood. The whole blood is treated with anticoagulant and incubated with fluorescent antibody, the whole blood is added with the RBC Lysis/Fixation Solution to lyse excess red blood cells for FCM. When added to the whole blood, antibodies bind to specific surface antigen on leukocyte. After treatment with 1x RBC Lysis/Fixation Working solution, the erythrocytes are lysed under mild hypotonic conditions, keeping intact viability of white blood cells.

Instructions

10x RBC Lysis/Fixation Solution is concentrated, dilute with ddH2O to 1x working solution before use. For example: take 1 mL 10x RBC Lysis/Fixation Solution, add to 9 mL ddH2O.The mixture is 1xRBC Lysis/Fixation Working Solution.

1. Add 100 µL of whole blood to a FCM tube.
2. Add appropriate volume of FCM antibody to the tube (refer to the antibody manual).
3. Vortex gently and incubate at 4°C in the dark for 30 min.
4. Add 2 mL of 1xRBC Lysis/Fixation Working Solution.
5. Vortex gently and incubate at RT in the dark for 10 min.
6. Centrifuge at 500 g for 5 min
7. Add 2 mL PBS to resuspend the cells. Centrifuge at 500 g for 5 min. Discard the supernatant.
8. Add 0.5 mL Cell Staining Buffer to resuspend the cells.
9. Analyze the cells immediately with proper machine settings.

Storage

Store at 2-8°C. Avoid of freezing. Valid for 12 months.

Cautions

1. Use EDTA as the anticoagulant instead of heparin.
2. Red blood cells may show in samples with nucleated red blood cells (such as pediatric samples) for the solution lyzes only enucleated red blood cells.
3. If the FCM antibodies react with serum immunoglobulins, blood samples should be washed with 1xPBS or physiological saline prior to staining and lysing.
4. This solution contains fixative which may damage fluorescence of tandem dyes such as APC/cyanine7.
5. This reagent is for research use only. For your safety and health, please wear the lab coat and disposable gloves before the experiments.