Could someone send me a good protocol to evaluate markers from mesenchymal stem cells by flow cytometry? if we have to fix the cells before the protocol or not, incubate with fbs or if we have an alternative for supplement, etc.
Thanks a lot!
Do you need the markers or just the protocol?
The protocol is pretty straight forward:
- detach cells (I prefer accutase)
- wash cells and resupend it in stain buffer (PBS, 2% FBS)
- use a cell concentration of 0.5-1.107 cells/mL
- add to each tube 100uL cell solution and antibodies
- incubate tubes in the dark for 30 minutes (May be done on ice or at room temp)
- wash the cells twice with Stain Buffer
- resuspend at 300-500 µl Stain Buffer
- analyze cells on flow cytometer right after or fix cells and analyze up to 48h later
If you need the markers
- CD90pos CD44pos CD105pos CD73pos
- CD34neg CD11bneg CD19neg CD45neg HLA-DRneg
If you don't have the antibody pannel yet, I recommend BD Stemflow™ Human MSC Analysis Kit.
In addition to the mesenchymal markers as described by Andre, it is also better to select the sca1pos cells to select the subset of stem cell marker positive cells from the mesenchymal cells.
Are you sure about Sca1? I always thought that only murine mSC expressed Sca1, I didn't kown that a human homologue was discovered.
I'll evaluate the markers that Andre described above, based on ISCT position.
Andre and Muruganandan, thanks!
Hello
I agree with Andre Fernandes ,and this other markers you can use CD166, CD90, CD44, CD29, CD73, and CD9
Good Luck
https://www.rndsystems.com/resources/articles/markers-and-methods-verify-mesenchymal-stem-cell-identity-potency-and-quality
http://www.bdbiosciences.com/documents/StemCell-AN-Multipara-Flow-Cyto-Analysis.pdf
https://www.rndsystems.com/products/multi-color-flow-cytometry-kits-mesenchymal-stem-cell-verification
http://www.ncbi.nlm.nih.gov/pubmed/23027703
http://cytobase.montp.inserm.fr/ftp/biblio/Rare%20Events%20Flow%20Cytometry%20Protocols%20Preprint.pdf
http://www.jove.com/video/51999/isolation-human-mesenchymal-stem-cells-their-cultivation-on-porous
In my understanding CD34 in human is homologous to Sca1 in murine. ISCT has adapted CD34 as a positive biomarker for human Mesenchymal stem cells since 2013. Our data shown the CD34 biomarker expressed in non-cultured MSCs, in bone marrow as in other human solid tissues, and gradually loss the expression after the in-vitro cell culture.
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