[Cytometry] bone marrow sorting and freezing
Tim_Bushnell at URMC.Rochester.edu
Thu Jan 19 20:50:37 EST 2012
To paraphrase Elizabeth Lane, "some say yes, and some say no .... I tend to agree"
I recommend lysis of RBCs and have had great success over the years. However, it all depends on the downstream application and if you can threshold the RBCs out.
Aut viam inveniam aut faciam
Sent from my iPad
On Jan 19, 2012, at 8:11 PM, "Guzik, Lynda" <GuziLJ at ccm.upmc.edu> wrote:
> Hello All,
> I could use some advise. I have a post-doc who would like to sort human fetal bone marrow for cd34+ cells. In talking with her, she is reluctant to lyse the rbc's as she is afraid it will kill her cells - if not the actual lysis followed by sorting, then the freeze/thaw process she uses (10% DMSO, 20% FBS) for long-term storage of the product. In the past, she has done several bone marrow sorts using magnetic columns and complained about poor recovery from the columns. (I do not know if she lyses the rbc's prior to loading cells on the column - probably not) My argument is that without lysing the rbc's, we will still have poor sort recovery due to conflicts, even with raising the fsc threshold and setting a second threshold triggering on the cd45 fluorescence. I am running a FACSAria II. The exp't plan is to stain with CD45 FITC, CD34 APC and PI for viability and sort using the 85u nozzle.
> What's the consensus of opinion? RBC lysis or not? Also, does anyone have any additional suggestions to increase recovery and viability, both from the sort and from her long-term storage?
> Thank you!
> Lynda Guzik
> McGowan Institute
> Bridgeside Point II - Rm334
> 450 Technology Dr
> Pittsburgh, PA 15219
> guzilj at upmc.edu<mailto:lguzilj at upmc.edu>
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