Mouse Erythrocyte Lysing Kit

Refer to the product datasheet for complete product details.

Briefly, erythrocytes can be lysed in splenocyte preparations using the following procedure:

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

View Graphic Procedure

 

The Mouse Erythrocyte Lysing Kit (Catalog # WL2000) contains the following reagents for the lysis of erythrocytes in splenocyte preparations:

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

This kit contains sufficient reagents to process 12.5 x 10⁹ splenocytes.

• 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 a single cell suspension of mononuclear cells from a mouse spleen with antibody or antibodies (if performing flow cytometry).
2. Wash the cells once with Hanks’ BSS + 10% serum.
 

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3. Disrupt the cell pellet by “racking” the tube.
4. Add 2 mL 1X M-Lyse Buffer per spleen processed.
5. Incubate the cells at room temperature until lysis is complete (10 minutes).
 

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

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8. Fix the cells with 100 uL 10X Fixative Concentrate if flow cytometry analysis will be delayed more than one hour.
9. Or
10. 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.


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

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