Hi all,
I am sorting small number of cells (10,000-50,000)in 1.5 ml tubes using FACS. I am unable to see pellet of cells in the tube. I understand that it is difficult to observe pellet with such a small number of cells, is there any way to visualize pellet? Is there any dye or beads available that can be used along with FACS sorted cells so that location of cell pellet in tubes can be visualized?
The downstream process which I intend to do with these facs sorted cells is RNA isolation.
Hello Arshed.
You do not have to see the cell pellet. Use 2 mg BSA/PBS-DEPC treated for the whole procedure of ICC (If you have not used this, cells have already made cell clumps). One of the major problems to lose sorted cells is non-specific adhesiveness of the cells to the microscopic surfaces of tube walls looking like Grand Canyon Valley. What you have to do is not to lose the sorted cells in the walls of tube. Add 1 ml of 2 mg BSA/PBS-DEPC treated to the sorted tube as a carrier and release the cells from the walls. If the total volume of the fluid becomes larger than 1.5 ml, divide the sorted tube into two tubes to facilitate the total volume. Gentle votex and brief spinning down at 1,000rpm for 5 min. Discard upper fluid leaving 50 ul containing cell pellets. Start RNA isolation.
Best wishes.
Lee is right, yours cells need to be in suspension, BSA will help avoiding clumping of cells
Hi Sang Ho Lee ,
thank you very much for your response. By 2 mg BSA/PBS you mean 2% BSA in PBS?
thenks,
Hi Arshed,
Just wondering if there is a specific reason why you would like to visualize the pellet. I am not aware of any dye or beads that you could use for this purpose. If you stain the cells with a dye for visualization, it usually remains in the cells and if you use beads, you will have to find a way to remove the beads after. In both cases, the additional manipulations on top of the stress induced during sorting could have an impact on gene expression. Whether or not this is a problem would depend on the genes you intend to look at.
If you would just like to see a pellet, you could try transferring your sorted cells into a small eppendorf tube and spinning in down. I used to do this whenever I was unsure where the pellet was and had to be really careful not to lose it. When you spin the eppendorf tubes in micro-centrifuges, you can direct the opening of the cap towards the center of the rotor and I think, your pellet settles on the opposite side (It has been a while since I did this and you might want to test it out first with a large number of cells where you can actually see a pellet just to be sure that it is the opposite side). Hope this helps.
Hello Arshed.
Sorry.
2 mg BSA/ml PBS. (Wait sprinkled BSA on top of PBS-DEPC treated to be dissolved to go-down). Millipore it. Store at 4C.
Regards.
Thanks Julie for your help. I will try to do trial centrifuge with large number of cells to see where in the tube I can see the pellet.
Hi Arshed.
Do not be greedy in the number of cells. This is very reason that you make clumps during cell collection in a small volume of fluid.
The followings are what I answered in a similar topic; (https://www.researchgate.net/post/How_to_increase_sorting_efficiency_with_BD_FACSAria_sorter)
Cell clamping is one of most frequent enemies you come across. Everyone in the lab did it, I recall. However, keeping the followings it never be a problem;
"Never be greedy about your cell concentration (use minimum), use of 1~2 mg BSA in all the procedure, brief spinning-down (very low, quick spinning) to collect the washed cells from immunostaining (needs relatively higher speed), gentle dispersion, not too long wait for FACS (prepare cells immediate before FACS), use of ice for cell stand-still even for a moment (this will depolymerize cytockelton in live cells), 0.26% NH4Cl for 20 min to block sticky aldehyde groups immediately after fixing cells in paraformaldehyde.
Best wishes.
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