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The RayBiotech 10X Whole Blood Lysing Solution is commonly used for lysing red blood cells following direct immunofluorescence staining of human peripheral blood cells with monoclonal antibodies prior to flow cytometric analysis. It is easy and convenient to use, and a perfect tool for immunophenotyping of human peripheral whole blood by minimizing the contamination of unlysed red blood cells.

Note: This lysing solution doesn’t contain fixative reagent.

• Flow Cytometer
• Fluorochrome-conjugated monoclonal antibodies to human leucocyte antigens
• 1.5 mL microcentrifuge tubes
• 12 × 75-mm tubes
• Distilled or deionized water
• Precision pipettes to deliver 2 μl to 1 mL volumes
• Centrifuge
• Vortex mixer

1. Dilute the RayBio? 10X Whole Blood Lysing Solution 10-fold with de-ionized H2O to prepare a 1X Lysing Solution.
2. Prepare an appropriate volume of a fluorochrome-conjugated monoclonal Antibody Cocktail in a 1.5 mL tube. Vortex thoroughly.
3. Label each 12 × 75-mm tube with the sample identification number and add 50 μL of well-mixed, anticoagulated whole blood from each sample into the bottom of the tube.
4. Pipette an appropriate amount of Antibody Cocktail from Step 2 above into the bottom of each tube and vortex gently to mix.
5. Incubate for 20 minutes in the dark at room temperature (20°C–25°C).
6. Add 1 mL of 1X Lysing Solution from Step 1 to each tube and vortex gently to mix.
7. Incubate for 20 minutes in the dark at room temperature (20°C–25°C).
8. Centrifuge the samples at 500 x g for 5 minutes and discard the supernatant without disturbing the pellet.
9. Add 0.5 mL of 1X Lysing Solution from Step 1 to each tube. The sample is now ready to be analyzed on a flow cytometer.