I am trying to isolate adipose tissue macrophages using FACS and am finding that I have a lot of debris. I have seen a few people recommend the use of a BSA cushion to eliminate debris following tissue dissociation, but I can't seem to find a complete protocol for doing this. Is the BSA-rich layer on the bottom? Is there a layer of media/other suspension fluid on the top? Does anyone have a protocol that they would be willing to share?
hi
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718226/
If you believe "BSA "created back ground or as debris in FACS analysis, you can use serum-free medium such as "SFM" thermo or EX-CELL® sigma . In addition, before staining the macrophages you should blocking Fc receptors with commercial Fc blocking reagents .
May God be with you. Amen.
Hi Reza, I appreciate you answering, although I think there may have been a misunderstanding. I am not concerned about background during sorting caused by BSA, I am trying to get rid of debris in my samples before sorting using a "BSA cushion" to generate a density difference (similar in principle to Lymphoprep, Ficoll, or Percoll). I was hoping that someone had experience with preparation of a BSA cushion, particularly with macrophages, to recommend a specific concentration and give me an idea of what to expect.
How about this?
Digest your adipose tissue with collagenase at 37C until it looks fairly well disaggregated. Spin 5 min at 700 x g. Top layer will be pure lipid, second layer adipocytes. Get rid of both of these. Resuspend the rest in PBS or medium. Large fragments of the stromovascular portion will settle to the bottom of the tube under gravity and should be avoided. The rest can be carefully aspirated and applied to a Ficoll cushion. Cells at the interface will be at least enriched in the macrophages you will be sorting. I see no virtue in using BSA as a cushion.
Have a nice day (as they say at MacDonald's)!!
Simon
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