HI, I used FACS (BD Aria III) for cell sorting.
I tested FACS cell sorting for in vivo cells isolation.
I used Raw264.7 cells with stained anti-CD11b-PE and anti-CD45-APC.
Cells were well stained both two antibodies.
I sorted (CD11b+, CD45+) total 720,000 cells into 15 mL tube (I filled 12 mL PBS before cell sorting). Cells were harvested by centrifugation and than I counted cells using hematocytometer.
Unexpectedly, I confirm 185,600 cells (25% yield)....
Dose it normal situation?
If it is not normal situation, how many cells can collect through comparing with interest gating cells number in display.
Additional info.
*Stream break off: normal
Thanks
JW Lee
Sounds like you're not losing them during sorting. Your sorter doesn't make up numbers when the cells aren't there. Your story is telling me that you're losing them during centrifugation. You should do another count between sort and centrifuge steps to reveal it.
Yes this is normal. It's extremely difficult to centrifuge small numbers of cells without large losses. The best way to do it is to leave a considerable amount of liquid in the tube, above the pellet, like 100 ul. And even then, pipette the supernatant off with a low-flow constant suction device rather than manual pipette, extremely carefully.
Hey,
Try to sort your cells into DMEM supplemented with FBS (your usual culture medium) instead of just PBS.
And as John already mentioned, most probably you are losing your cells after sorting, potentially during centrifugation, I would centrifuge at low rpm for at least 20 minutes. Yes, some cell loss is expected but not to the point of losing 3 quarters of your cells. Best of luck
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