We are working with adipose-derived stem cell differenciated into adipose cell. We are having trouble with them to perform a cytometry because of the lipid contents. Do you know how we can perform a cytometry and get rid of the lipids?
Adipose cells (which means they have triglycerides) are not appropriated for flow cytometry because they easily break. One possibility is may be to fix the adipose cells (I did not try), of course you will not be able to used them in culture, it may work for small size adipose cells. I am afraid that it will not work for large size adipose cells which may remain fragile despite the fixation.
I did use to fix them with acetone at -80 for 5' for immunofluorence. It did work perfectly for that technique because at the same time acetone dissolve the lipid, permeabilize the cells and fix them.They kept their shape and react with the abs. May be you can try the same method, is very easy and fast.
I have assessed perilipin expression during adipose derived stem cells adipogenic differentiation using flow cytometry. I have harvested the cells from the culture plates by trypsin-EDTA treatment. 1.4 x 10
If you have difficulties in performing cytometry, using morphological observation, gene expression analysis and cytological staining can confirm an adipogenic differentiation of ASCs.
it is not a good idea to analyse by flow cytometry after the cells fully differentiate (after about 2 weeks) into adipocytes. As adipose MSCs are very effficing in adipogenic differetiation, it only take 2-3 days to induce changes. at this stages although morphology of cells changes but there are no lipid droplits at this stage. So i will recommend to analysis at this stage.
Mahmood S Choudhery
You put them in culture without feeding them, they are going to loose their lipides
I'm working with Mesenchymal stem cells derived from stromal vascular fraction obtained following enzymatic digestion of lyposuction material. Really it is very difficult to analyzy by flow cytometry this material, I was surprised that when I analyzed the same material following cryopreservation in liquid nitrogen and DMSO 10 % the flow cytometry analysis was very clea and the cells alive. I cultured them without any problem. I hope it will help you
good luck
Paola Iudicone
I'm working with Mesenchymal stem cells derived from fat, it is difficult to analyzy by flow cytometry, but using liquid nitrogen and DMSO 10 % , was great.
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