I need to see the effect of negative depletion of subpopulations expressing different surface markers on my target heterogeneous cell population. There is around 20 markers, for each of them depletion will be done individually in a separate experiment. Target cells do not like sorting, and cell numbers for the experiment are going to be low as these are isolated primary cells. For depletion I will have to use FACS antibodies, probably PE conjugated. Can you recommend methods of depletion other than sorting?
Using MACS columns is also not very practical as cell numbers are very low and they seem to stick to the column a lot. Also due to a high number of samples (20 markers and repeats) using MACS would be very expensive.
Can you do something like complement lysis with FACS antibodies?
You mentioned that your target cell population does not like sorting. So I am assuming that you just need to isolate one specific cell population, but want to do it by sequential depletion of other cell populations. And that you would have 20 rounds of depletion using one marker in each round?
This would be heavily labor intensive. The amount of time you would spend taking the cells through all the depletion processes and also the number of manipulations you are subjecting them to would surely have an impact (mostly negative) on whatever analyses you are planning to do with your isolated target cell population.
The best strategy would be to isolate your target cell population using a selection method (positive or negative). There are kits out there for almost every conceivable cell subset. If column based methods (MACS) aren't working for you, use a bead based method. StemCell Technologies have really great kits (I have experience using several ones over the years). This would be more expensive but the alternative would be to use strategies that could compromise the quality of the data you generate in the end.
No, I guess I expressed it in a wrong way (edited). Depletions are to be done for each marker individually. So it is going to be 20 separate depletions.
I was thinking about EasySep but not sure how well it would work with =
Thanks for clarifying.
If I understand you correctly, you want to deplete individual cell subsets from your mixed cell population and see what the impact is, when you remove a particular subset (on your target cell population). Also, you would like to avoid sorting. Please correct me if I am wrong.
In this case, what I would recommend is again to use bead based separation. Label the cell subset you want to remove with antibodies conjugated to PE. You then remove the PE labelled cells using anti-PE microbeads/magnetic particles (anti-PE microbeads from Miltenyi/ EasySep PE positive selection kit from SCT/anti-PE Magnetic particles from BD). I hope this strategy makes more sense.
Yes, that is exactly what I am trying to do.
I just saw StemCell has a bead-based depletion kit in a plate format, maybe I will try that one with anti-PE beads.
Thank you for the answer and recommendation.
Hello
Yes, i agree with Julie Joseph; the best choice for you is using bead-based separation.
Good Luck