Hi,
I plan to sacrifice 5 mice in one day, and I want to take some organs. I’ll also collect blood and bone marrow for flow cytometry. But I don’t know how to arrange that properly because all sarcificing will take around 3-4 hours. Can I collect blood in EDTA tubes and put femur/tibia in DMEM or PBS, and then put them on ice until finishing all mice?
Here’re my plans.
Plan A:
1. Collect blood in EDTA tubes -> put on ice
2. Take organs (e.g. spleen, liver..)
3. Remove muscle*** around femur/tibia and put in DMEM? or PBS? on ice
4. Sacrificing next mouse …
***Can I put whole leg with skin and muscle in DMEM or PBS at step 3?
Plan B:
1. collect blood in EDTA tube -> RBC lysis -> add PBS -> centrifuge then discard supernatant -> add FACS buffer and keep on ice (one person do that)
2. Take organs (another person continue…)
3. Remove muscle around femur/tibia and put in DMEM? or PBS? on ice***
4. Sacrificing next mouse …
*** I’m not sure if I can just put femur/tibia on ice and then continue to do bone marrow isolation after all sacrifing.
Could you please give some suggestions?
Many thanks.
Hi,
I think it is best to first collect blood in an EDTA tube and keep it on ice. Most cells from peripheral blood in EDTA tubes are quite stable. Afterward, remove the organs and give them to another person; I think this step can be done quickly. Then, collect stem cells from the femur and tibia immediately. Hematopoietic stem cells lose viability quickly and are very sensitive. When the stemcells are in the media continue with the next mouse.
Alternatively, you can get a third person involved to already process the EDTA blood, but I don't think it is necessary.
Good luck.
Tessa M Alofs Hi Tessa, should I put stem cells isolated from bone marrow in the DMEM with or without FBS? Which one is better？Many thanks.
I would add 10% FBS. But we always used RPMI 1640 media. I don't know the exact difference. Our protocol was similair to this one: https://www.thermofisher.com/nl/en/home/life-science/cell-analysis/cell-analysis-learning-center/immunology-at-work/immunology-protocols/mouse-bone-marrow-cell-isolation.html
Might help you,
Good luck again.
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