Hi everyone,
I’m following a protocol to flush bone marrow from mouse femurs and tibias, but I consistently get little to zero cell yield. After RBC lysis and centrifugation, I don’t see a white pellet, and no viable cells are detected during counting.
Here’s my flushing procedure:
- I use 2 mL of PBS/2% FBS for 2 legs.
- Femur: I cut the ends using curved scissors, insert a 23G needle, and flush both sides until the bone turns white.
- Tibia: I shave the knee end, flush one side multiple times, and ensure the bone is white.
- I spin at 300xg for 6 min
- Afterwards I add 4 mL of 1x RBC lysis buffer to incubate for 5 mins at room temp. This is then topped with 8mL of PBS/2%FBS and spin again at 300xg for 6 min. ( I tend to not see a white pellet after this step)
Despite this, the yield is zero. Am I not using enough PBS, or am I something I’m missing else? Has anyone faced a similar issue or have tips to improve the process? I've attached my protocol as well for better clarification.
Any insights or advice would be greatly appreciated!
Thank you!
When you say, the yield is zero, does that means there are no cells (including dead ones)? Do you see the red pulp after the bones are flushed? Check the cell count and viability at this point before adding the RBC lysis buffer. Your RBC lysis solution could be too harsh.
Bindu Parachalil Gopalan: When I flush out the bone marrow and spin it, I do see a pellet. It's after the RBC lysis I do not see any more pellets. In the end, sometimes I do see a few dead cells for some mice and for others, nothing at all. I will check the cell count and viability before adding the RBC lysis. Thank you!
You may try using this method for red blood cell lysis: resuspend the cell pellet in 20 ml of 0.2% NaCl for approximately 20 sec followed by addition of 20 ml of 1.6% NaCl. (Critical: Do not exceed 20-30 sec of hypotonic lysis to avoid bone marrow cell death. The use of hypotonic NaCl for lysis is
recommended over ACK lysing buffer because the latter has the potential to activate the neutrophils). This is an excellent method. See the attached paper.
Refer this paper:
URL: http://www.jove.com/video/50586
DOI: doi:10.3791/50586
Hi Floriane,
As Bindu Parachalil Gopalan said the problems you encounter might be because the RBC lysis is too harsh and you might also lyse the nucleated cells from BM together with the RBC. You can check the total number of nucleated cells in your BM by treating your raw BM suspension with a solution of 3% acetic acid and 0.25% methylene blue that will lyse the RBC and the membranes of the nucleated cells. Mix 10 ul solution with 10 ul BM suspension and count the obtained nuclei on a haemocytometer to check if the numbers match with the literature.
Also, from the protocol you described it is not clear how you disperse the cells from the BM clusters collected from the bones. Needles to say that if you do not do it right you might have downstream problems.
Good luck!
Hey everyone! Thank you for your feedback! I’ve been troubleshooting my protocol and ive been getting great cell yield now!
For a bit of context, I’ve switched to bone marrow isolation by centrifugation.
As for flushing, my little to no cell yield was most likely due to the potency of the RBC lysis buffer.
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