at the moment I'm trying to optimize my protocol for the isolation of stromal vascular fraction from human adipose tissue for flow cytometry measurements. According to my protocol I first wash the tissue with PBS, cut it into 1-2 mm pieces and digest with 1 mg/ml collagenase in krebs-ringer-bicarbonate buffer for 40 minutes at 37°C. After the digestion I filtrate the cells first through 100 µm and 40 µm (this step takes longer if I have more tissue), wash with ice cold krb buffer twice the initial volume and do two centrifugation steps.
I would have a few questions now:
- is the collagenase reaction stopped by adding the ice-cold krb-buffer
- how big is the effect of how long the tissues were in PBS before cutting (what is the maximum)
- what could be the most important cause of variation (cutting, collagenase duration?) and how I could minimize the variation between the samples and treat them as equally as possible (which steps are important to be kept exactly the same, if I have more tissue and the time factor starts to play a role).
Looking forward to any answer...