# Isolating stromal vascular fraction from human adipose tissue

**URL:** <https://scientistsolutions.discourse.group/t/isolating-stromal-vascular-fraction-from-human-adipose-tissue/624>\
**Category:** Cellular & Molecular Immunology\
**Created:** [January 6, 2025, 12:53pm UTC](https://scientistsolutions.discourse.group/t/isolating-stromal-vascular-fraction-from-human-adipose-tissue/624 "2025-01-06T12:53:24Z")\
**Posts on this page:** 1\
**Page:** 1

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**Author:** ![Sarahcanon2025](https://avatars.discourse-cdn.com/v4/letter/s/f04885/32.png) [@Sarahcanon2025](https://scientistsolutions.discourse.group/u/Sarahcanon2025)\
**Post date:** [January 6, 2025, 12:53pm UTC](https://scientistsolutions.discourse.group/t/isolating-stromal-vascular-fraction-from-human-adipose-tissue/624/1 "2025-01-06T12:53:24Z")

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Hello 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 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).

Thanks