I sorted bm LSK cells and got around 15,000 cells from 4 isolated bones (femur + tibia). We are interested in LSK CD150+ CD48- subfractions as well as CMPs, GMPs, MEPs, afterwards we want to characterize them functionally (colony forming assays, RNA isolation, cell cycle status, apotposis,). Since the progenitor/stem cell fractions are very small, I don´t have any idea how to manage them. I have already tried different things on FACS (cell cycle, apoptosis,), but the combination with surface marker staining has not worked well so far - it would be very helpful if somebody could give me advice. Thanks
For sorting stem cells, you could try magnetics bead to select your cells. The main advantage is the time of selection is lower than in FACS sorting, arround few minutes. The more efficient method is to use automated system like the "automacs". You obtain a very good purity.
I suggere a postive selection and not a depletion of others cells due to the low cell precentage. One problem is that the cell keep the antibody and the bead. It could be a problem: the cells might be activated by the presence of the antibody/bead.
Mouse bone marrow cells are small in size and so there is always a higher chance that you may lose out on the important cells of interest when you sort them on a FACS. It is always a better option to sort the cells using magnetic beads from Miltenyi Biotech. This gives a good yield and a pure population. You dont lose out on cells while washings also. In addition, I would suggest that you pool the cells from multiple bone marrow samples. This will give a better result also as pooling always helps.
More time incubation on ice, more antibody, probably would do you some good
Yes I agree with Shabari and Stephane, I use the magnetic beads as well.
Thanks for your advice but which cells are you sorting (by MACS I guess) - this is not possible for me because I characterize my population with 6 - 7 surface marker or is there another option??
For sorting I agree with the above posters that enrichment (like c-kit enrichment or lineage depletion) is a good choice. For analysis however I would not go for the enrichment option. Usually people quantify based on % of total BM cells, and that is not possible to do should you use enrichment. Since you are apparently going for post sort analysis, do enrichment. I do not see the problem that you have 6-7 surface markers and enrichment, as you metioned above. Either use lineage repletion with biotinylated antibodies, or c-kit enrichment using anti APC, FITC beads or whatever fluorochrome your c-kit AB is.
The other questions are difficult to answer since I do not know what equipment you have. Normally we run up to 8 colors on the flow, but all depends on the equipment you have. If you "only" use LSK CD150 CD48, then you should be able to do cell cycle as well, either directly by analyzing while sorting, or on the sorted fractions.
The numbers you got, 15.000, is not too bad at all, if you add the pelvic bones and spine you would increase the numbers, but of course at the same time need more time. This is all tricky business, and you will need a lot of time to optimize the conditions. If your yield is low, I would recommend RNA extration using picopure. This is working well in our hands from a few hundred of cells. If you are interesting in colony assays, sort in media, take out an aliquote for plating/cell cycle or whatever analysis you want to do, then spin and resuspend the pellet in picopure for RNA extraction.
As a general rule, the more surface markers you use, the purer your cells but the lower the yield. We had pretty good luck with sorting these cells BUT I have to admit, much depends upon the skills and advice of the FACs operator who is the most important variable. For us, working with a skilled person at FACs has vastly improved our outcomes.
The humerus is another source of marrow that can be easier to dissect than pelvis or spine, although those are good sources as mentioned above. It is still large enough to flush and increase yield from a single mouse if you are not able to pool mice. I found it only added a few minutes to the dissection and a few minutes to the flush step.
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