Hi all, 

I am working with single cell suspension of kidneys by enzymatic digestion for the first time. I did following steps.

1) chopped the kidneys, passed through 100uM cell strainer  and digested the tissue in Collagenase-Dispase and DNAse combination at RT for 30 min, then stopped the reaction by adding wash buffer with EDTA 2mM.

2)  passed the cells through the 70 uM  cell strainer.

3) centrifuged 500 xg for 20 min at 4^0C ( I tried 300xg for 10 min first but very few cell at the bottom) 

4) Resuspended in red cell lysis buffer (ACK lysis buffer containing 8.29 g NH4Cl, 1g KHCO3, 37.2 mg Na2-EDTA,pH to 7.2-7.4) for 5min and filtered through the 40uM strainer. 

5) centrifuged 500 xg for 20 min 4^0C ( tried 300xg for 10 min first but very few cell at the bottom)

6) resuspended in 1mL FACs buffer and counted.

The cell count was very low 3X 10^4 cells/ ml

I was expecting lot of cells than these. Can anybody please suggest what steps were wrong or need to improve? Also, should the centrifugation be done at room temp  instead of 4^0C.

Thanks in advance.