Hey everyone, I am trying to isolate mesechymal stem cells from human umbilical cord, and I am looking for the expression levels of CD44, CD90, CD34, and CD45 at 3rd, 5th, and 7th passages for characterization. Here my question; expression level of CD45 is about 3-4 percent at 3rd passages of my 1st and 2nd samples,then after a while, the level of CD45 expression for the 3rd, 5th and 7th passages of the 3rd, 2nd and 1st samples respectively got increased to 20 percent level, and also the pozitivity for CD34 got increased from 0,5 to 1,5 - 5 percent.. I am sure I followed the same protocol as I did before for the staining cells, but the analysis is being done by the technician responsible for the FACs machine.. and I am not competent at using this FACs machine.. Does anyone has any idea about this situation, and if there is one studying MSC and using the BD FACsCanto II, can give voltage values applying for the above mentioned markers..And finally my FSC-SSC graphs for some of samples are so scattered, what may cause this stiuation ? thank you...
This has happened 2 times. It is possible it is real. I have a friend trying to look MSCs. His panel might be different. The tech very likely is not the sourse of the problem. However, do you have single color controls to make sure that your gates are set properly? Improper gating may be responsible foer the changes observed.
I did change gating area, and the results did not change.. However I noticed that during the staining procedure, I was applying the vortex to resuspend the cells in the FACs tube, but today I have just applied pipetting to resuspend the cells, and my results get improved to %2-4 percent. Actually vortexing should correct the scattered graph problem but it also reduce the positivity of the CD45 and CD34. thats great but now I am questioning that vortexing the cells during the staining is resulting in false positivity ? (And what else can I do to improve this 2-4 percent CD45 positivity to less than 1 percent ? )
This is my colleague's panel-
huCD44 PE;huCD90 APC ;huCD45 Percp cy5.5;huCD29 FITC;huCD105 Pacific blue
Obviously, the colors can always be manipulated to fit your instrument and your preferences. I highly doubt that vortexing the samples will cause the changes observed. The scatter has to do more with voltage and perhaps sample variability. Depending on you isolation method, you may want to make sure your cells are healthy by including a live/dead gate. Good luck.
The basic question I have is how you isolate the MSC from cord blood. My guess is that you are carrying a small number of CD34+ HSC along in the preparation, and they are seeding on the MSC and growing out slowly, giving rise to CD45+ cells. Another possiblilty is the CD45+ cells are predominantly macrophages. Macrophages express Fc receptors and will bind antibodies. Do you include any FcR blocking reagents [either human IgG or antibodies to human FcR]?.
Hi serby Oskan
Probably you have non pure cells. Please continue expansion of UCMSCs for 1 or 2 passage and discard unattached cells 4-5 h after new passage. when confluency of cell reach to 80% wash your flask 2-3 time with PBS and then remove your cells with tripsin.
- Badges
- Science topic