Human Erythrocyte Lysing Kit

Refer to the product datasheet for complete product details.

Briefly, erythrocytes can be lysed in whole blood using the following procedure:

• Stain whole blood or an enriched mononuclear cell preparation with antibody
• Add H-Lyse Buffer
• Wash cells with Wash Buffer
• Fix cells for flow cytometry with Fixative

View Graphic Procedure

 

The Human Erythrocyte Lysing Kit contains the following reagents for the controlled lysis of erythrocytes in whole blood:

• H-Lyse Buffer Concentrate (10X)
• Wash Buffer Concentrate (10X)
• Fixative Concentrate (10X)

This kit contains sufficient reagents to process 250 mL of whole blood.

• Ficoll-Hypaque™
• Sterile distilled or deionized water
• Sterile centrifuge tubes
• Benchtop centrifuge
• Pipettes and sterile pipette tips

R&D Systems Protocol for the Lysis of Erythrocytes Using the Mouse Erythrocyte Lysing Kit

1. Stain 100 uL of whole blood with antibody or antibodies (if performing flow cytometry).
 

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2. Vortex cell pellet vigorously.
3. Add 2 mL 1X H-Lyse Buffer.
4. Vortex vigorously.
5. Incubate the cells for 5-10 minutes at room temperature.
6. Pellet leukocytes using centrifugation.
 

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7. Wash the pelleted leukocytes with 2 mL 1X Wash Buffer.
8. Resuspend the cells in 1 mL 1X Wash Buffer.
 

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9. Fix the cells with 100 uL 10X Fixative Concentrate if flow cytometry analysis will be delayed more than one hour.
10. Or
11. Utilize leukocytes for alternate downstream applications
 

If the cell preparation will be further processed with a T cell enrichment column, complete these steps prior to treating cells with H-Lyse Buffer.


12. Prepare a single cell suspension of mononuclear cells.
13. Wash the cells with excess sterile PBS.
 

• If flow cytometric analysis of the cells will be delayed for more than 1 hour following erythrocyte lysis, the cells can be fixed at this time to stabilize them for later analysis. This step should be eliminated if cells are to be used for cell culture.
• Fixed cells should be stored at 2 °C to 8 °C until flow cytometric analysis. Although stained cells will be stable for up to 48 hours, we recommend that flow cytometric analysis be performed as soon as possible.