I am trying to treat immortalized cells with human plasma of interest, but once the plasma concentration is more than 1% in the well, I am finding a thick gooey substance coating the bottom and sticking to the cells, causing the cells to get aspirated out when washing with PBS. Unfortunately I don't have access to human serum samples. Does anyone have any tips/tricks to prevent this type of cell loss?
Dear Jennie,
This happens often when using plasma or platelet lysates, as there is a conversion of fibrinogen into a fibrin gel. What you can do is to add 1 to 3 IU/ml (final concentration) of heparin, an anticoagulant, to the medium. Another approach would be to convert first the plasma into a serum by inducing coagulation by addition of 20-25 mM CaCl2 to the plasma, possibly in the presence of a surface activator like glass beads. You can then remove the fibrin clot by centrifugation and use the supernatant to treat the cells. I hope this solves your problem. Kind regards, Thierry
Thank you, Thierry. I will try the CaCl2 approach as the plasma was processed in sodium citrate. How long should I wait for a clot to form (or at which point if I don't see a clot would I consider it not working)? Do I have to have it at a specific temperature to work?
Thanks!!
Hi Jennie, you should do it at 20-30°C range, under mild stirring. With glass beads (or pieces of glass) the clot should form in 10-20 min, and in approximetely 30 min without glass. CaCl2 will counter-balance the anti-coagulant effect of sodium citrate, allowing the activation of the coagulation cascade, and ultimately the conversion of fibrinogen into insoluble fibrin. Good luck!! Thierry
- Badges
- Science topic