We are performing a whole blood lysis procedure on heparinized blood (3mL) to lyse RBCs and later remain with WBCs that we count and freeze down and later cryopreserved in liquid nitrogen for future flow cytometry experiments.
We lyse for 10minutes with 1X FACS Lyse buffer (BD). We included a washing step with 1X PBS, followed by centrifuging and resuspend the pellet in 2%FBS and PBS.
We later stain with trypan blue and do cell count for WBCs, however the viability is so poor, most of the cells are dead.
We have also tried without the washing step but the viability is still very poor.
What could be the problem? Any one who has got good viability of WBCs after RBC lysis using heparinized blood?
Please we need some answers, thanks.
Technically the RBC lysis buffer does not do anything to WBCs.
But, as you said you are having dead cells or degenerated cells, that occer due to change in pH of your RBC lysis buffer (It is freshly prepared) or the pH of Staining buffer (2% FBS+PBS).
The FACS buffer says it's only for use for lysing RBCs after staining. These are not designed to be used to lyse RBCs in live culture.
You should try ACK buffer. Although it may take at least two rounds. I usually use 5mL ACK / 1mL whole blood... But you will most likely have some residue and will need to resuspend in a smaller volume of ACK buffer to get rid of all the remaining RBCs.
@ Shen-An,
FACS lysing solution lyse erythrocytes under gentle hypotonic conditions while preserving the leukocytes. Please refer to the attachment .
Hello Rhoda,
I am sure you must have followed the protocol as per manufacturers instruction. But some of the steps you should recheck are:
- Did you dilute the lyses solution (1:10)?
- Did you neutralize (blood+Lyses solution) with excess of FACS buffer/1XPBS beforecentrifugation?
- Also, remember, do not spin the blood at high rcf (or g). Usually spinning cells at 250g for 5 minutes is enough. RBCs,dead cells, nuclei and organelles are lighter than live cells, and will not pellet at lower speed (unless you go on spinning for more time). You should get a cleaner sample post-centrifugation.
- How much time did you incubate the cells in Lyses solution? If you incubate for more time, you will see increase in cell death (other than erythrocytes). Same is true with ACK-lyses buffer.
Trouble shoot suggestion:
Hope this helps, Good luck,
Best regards
Savita
Technically the RBC lysis buffer does not do anything to WBCs.
But, as you said you are having dead cells or degenerated cells, that occer due to change in pH of your RBC lysis buffer (It is freshly prepared) or the pH of Staining buffer (2% FBS+PBS).
Thank you so much for all your responses.
Savita;
To answer your questions;
To note, I spin the sample first at 1700g for 10min and harvest plasma for storage. Then I further work with the remaining cells as below;
1. I diluted the FACs Lyse to 1X in a ratio of 1:10
2. Incubated the sample with 1X FACs lyse for 5 mins
3. I spin at 500g for 5mins.
4. Wash the cells in 1X PBS at the same speed as above
Regarding neutralization; I didn't do the step. How much of the excess FACs buffer and 1X PBS should I add? Do I have to incubate for this step? OR I spin after adding these two?
From the insert it says to incubate for 10mins in FACs Lyse, however my viability has been very low. Today I tried incubating for 5min and viability improved.
I will also try out your advised trouble shooting steps.
Thank you
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