Hello,
For our humanized mouse model we are isolating CD34+ cells from cord blood units using MACS kit. We found CD34+ purity about 98% after MACS enrichment. However, when frozen and thawed our samples were checked in another lab and the percent of CD34+ cells was way lower - just about 50% or so. I am guessing this might be because of a different instrument and the gating strategy used when cells were thawed.
Can somebody suggest an optimal gating strategy for enriched CD34+ cells from cord blood?
I am attaching flow cytometry plots from before and after freeze-thaw.
Any suggestions highly appreciated.
I am haematologist and working in the fields of human haematology.
Isolation in human can be found in;
https://www.stemcell.com/isolation-of-cd34-cells-from-human-cord-blood.html
https://cellculturedish.com/questions/what-is-your-advice-for-the-best-way-to-isolate-hematopoietic-stem-cells-from-cord-blood-and-remove-erythroblasts/
Article CD34+ AC133+ Cells Isolated from Cord Blood are Highly Enric...
Hello Danila - there could be a number of differences contributing to this. Like you said - different lab, different instrument, and different gating strategy. I would also double check antibody clones, and compare gates for the same positive & negative controls between your lab and theirs. But first, it is not clear from your attached data if the post-thaw samples were first gated on viable cells before gating for CD34. Your freshly isolated cells were likely close to 100% viable (am assuming some type of density media separation prior to MACS bead isolation which also removes dead cells), but post-thaw are very likely much less than that. CD34+ HPCs are especially vulnerable to dying during freeze (this can be very variable depending on technique, method, and original donor - I've seen anywhere from 30-95% viable post-thaw with primary HPCs). If this is the case here - cell death, plus no viability marker, then that could completely explain the change in %CD34+ between analyses.
Dear Lindsey Crawford and Abdulsalam Al-Ani thank you very much for your comments and information you shared. Indeed, the problem could be complex. Now I am questioning the freeze technique. It is quite possible that big loss of the cells is happening during freeze procedure which has to be revised I believe.
Thank you a lot!
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