Isolating stromal vascular fraction from human adipose tissue

Dear all,
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...
Thanks

 Yes, cold will reduce the kinetics of your enzyme reaction.

My guess is that the intial PBS stage has little effect other than to wash the sample, longer or shorter periods of time at this stage are likley not to make a difference (within reason).  

Your collagenase step is were I expect the variability.  Time, temperature, size and volume of tissue will all effect the outcome.  This is the part that it is hard to mechanize because an experienced person can eye-ball the progress and know if it is best to go a little longer or shorter.  To minimize this try to use the same amount of tissue (g tissue/ volume of collagenase).

Hi Carson,
thank you very much for your suggestions. I always use the same relation between weigth of tissue and collagenase solution (3ml / g tissue). From your answer I concluded that there is a variation in collagenase activity depending on the weight of the tissue (even if I always use the same relation between weight and volume?). Did I understand it right?

Thanks!