Adipocytes preparation for FACS (cells from tissue)

[lior]

I would appreciate if you could advise me regarding the preparation of adipocytes for FACS analysis.

Annexin V and propidium iodide can be used in FACS to discriminate quantitatively between apoptotic cells and necrotic cells. Annexin and PI will not penetrate into intact cells, while cells that will be stained by Annexin only indicate apoptotic cells, and cells that will be stained by both Annexin and PI indicate as necrotic or necrotic+apoptotic cells.

I would like to perform this assay following ultrasound treatments on pigs (in vivo) and recovery of adipocytes from the tissue using trypsin or other common methods. However, I am afraid that the trypsinization procedure itself will make holes in the membranes and increase the number of false positives. I am aware of the fact that some formation of membrane pores might be inevitable following trypsin treatment, but I believe that we could minimize this effect by optimizing the trypsin concentration and exposure time. Needless to say, an internal control will be included as an integral part of the study so that we could be able to distinguish between the effect of trypsinization on the membranes and the treatment effect.

1. Are you familiar with trypsinization of adipose tissue for FACS analysis (or for another application)?

2. Could you provide me with such a protocol? (Reaction times, concentrations, enzyme and buffer suppliers, etc.)?

3. What is the stop solution that should be used to stop the reaction?

4. Other tips are welcome.

Thanks in advance,
Lior

[Priscilla]

Trypsin Neutralizing solution (TNS) stops trypsin's reaction.

[LilKim]

I have used trypsin/collagenase solutions to make single cell suspensions... and the cells lived and were healthy. So, i would guess that the trypsin, when done properly will not damage the integrity of the cell membrane.

Also, anything containing serum will stop trypsin's action. So, if you treat the cells in trypsin... and after resuspend them in media (containing serum) the reaction should be stopped and the cells won't be damaged.

I think what you'd have to do is perform time trials to determine how exaclty to trypsin treat (ie. concentration and how-long) before stopping the reaction in media. And, then staining with PI and then maybe even simple microscopic analysis for red fluorscence will give you your answer (whether the trypsin damaged the cells.. and cause apoptosis/necrosis)

Good Luck!
- KIM

p.s. Unfortuantely, i do not have a trypsin/collagenase protocol now ... at the time that I did this work, I worked for a private company and the protocols were 'proprietary' ... and it's been years now, and I just don't remember... sorrry!