I previously attempted to lyse RBCs in mouse whole blood using ThermoFisher’s eBioscience 1X RBC Lysis Buffer, following the manufacturer’s protocol exactly - 10 mL of lysis buffer per 1 mL of blood, with occasional mixing and 5 mins incubation at RT. However, the lysis did not appear to be successful. Even after repeating the lysis step twice, the cell pellet remained bright red, suggesting incomplete lysis. This was further confirmed by microscopy.
I have now acquired BD Pharm Lyse Buffer and plan to retry RBC lysis using this reagent. Could anyone please advise on the optimal volume of BD Pharm Lyse buffer to use with mouse whole blood, along with the recommended incubation time and temperature?
Additionally, any insight into why the eBioscience RBC lysis may have failed would be greatly appreciated too.
Thank you in advance!
Hi Dear Dayangku Nordiyana B P Hassanel
Your issue with incomplete RBC lysis using eBioscience 1X RBC Lysis Buffer is not uncommon when working with mouse blood, which is often more resistant to lysis than human samples due to species-specific differences in RBC membrane composition and density.
Optimizing RBC Lysis Using BD Pharm Lyse:
BD Pharm Lyse Buffer is generally more effective with murine samples and commonly used in flow cytometry and immune cell profiling from mouse blood.
Recommended Protocol for Mouse Whole Blood:
- Dilution: Dilute mouse blood 1:10 with BD Pharm Lyse Buffer (i.e., 1 mL of blood + 9 mL of BD Pharm Lyse).
- Incubation: Incubate at room temperature (20–25 °C) for 10 minutes, with occasional gentle mixing (inverting or flicking every 2–3 minutes).
- Centrifugation: Spin at 300–500 × g for 5 minutes at room temperature.
- Supernatant Removal: Carefully aspirate the supernatant without disturbing the pellet.
- Wash: Resuspend the pellet in PBS or staining buffer and centrifuge again to remove residual lysing agent, which can be toxic or interfere with downstream assays.
✅ Tip: If RBC contamination persists, you may repeat the lysis once only briefly (1–2 minutes) to avoid leukocyte loss.
Why the eBioscience RBC Lysis May Have Failed:
Final Suggestion:
If BD Pharm Lyse still doesn’t yield satisfactory results, consider ACK Lysing Buffer (Ammonium-Chloride-Potassium), which is widely used and often more potent for mouse blood. Alternatively, hypotonic water lysis with brief exposure (20–30 seconds) followed by isotonic quenching can also work with care.
Be careful to adjust your experimental conditions to keep the pH between 7.1-7.4 and avoid light. Alternatively, you can extend your incubation time appropriately instead of repeating the cell lysis step twice, which risks damaging the cells. If optimizing the conditions does not solve your problem, it may mean that your kit has lost its effectiveness. After replacing the new kit, you can incubate for 5-10 minutes.
If you need further experimental consultation, you can contact our technical colleagues to apply for email or online technical guidance. For relevant product information, please refer to the following link page.
https://www.medchemexpress.com/inhibitor-kit/red-blood-cell-lysis-buffer.html
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