Hello

Kindly look at links here

Article Fluorescence-Activated Cell Sorting Analysis of Mitochondria...

http://www.renyi.hu/~stipsicz/skin/Flow_cytometry_book/Ch_17.pdf

Good Luck

Hi Ankur, we found that sorting in tubes pre-filled with FBS helps - in our case, it does not interfere with subsequent assays, but I don't know if this could apply to yours. Other research groups here also use FBS-containing culture medium (e.g., 20% FBS if the regular culture medium for the sorted cells uses 10%). Maybe it could be worth a try.

Thanks Lavinia, but I am planning to use these cells for RNA-seq experiment. I am not surer whether including FBS can cause any inappropriate signals as shown in some publications like this: https://www.nature.com/articles/srep31175.

Indeed, FBS would be a problem then. Also, most serum replacement products on the market wouldn't necessarily be RNA- or RNAse-free (actually, I don't know if any would even work for regular hepatocyte culture, i.e., if the hepatocytes wouldn't mind it). I wonder if you could use some magnetic selection, such as negative selection by CD45, which would likely be milder on the cells...

P.S. For better recovery and to avoid clumping during sorting (we are interested in lymphocytes at a reasonable purity) we also use a larger nozzle (85 rather than 70 micron), DNAse I pre-treatment and EDTA in the pre-sort buffer (not sure if you can do that).

Yes you are absolutely right! Magnetic selection would be better option. But hepatocytes are generally bigger ( upto 50 um) and it may cause a clogging in the magnetic beads. However we are planning a trial run soon using micro beads.

Regarding sorting, yes we are also using 85 um nozzle but I have not yet tested the DNAse-I pre-treatment and addition of EDTA in presorting buffer. What concentration and incubation time do you use for that ?