Hi John,

I have two suggestions to avoid this (which is not too surprising I think). Can you (a) use a higher concentration of fibrinogen, and (b) centrifuge the cells to remove the cell culture medium? Once you pelletize the cells, they could be resuspended by the fibrinogen solution (with or without CaCl2) and after mixing you add the thrombin at equal volume. You will get a fibrin gel seeded with cells, if this is what you wish to get. Kind regards, Thierry

Hi Thierry Burnouf ,

Thank you, I'll give this a try! If I may ask, what does increasing fibrinogen do? I'm currently using 3 mg/ml because I performed a previous experiment with 3 and 6 mg/ml fibrin and the cells seemed to spread better at 3 mg/ml!

Increasing fibrinogen concentration will give you a fibrin clot with a stronger strength, more dense honeycomb structure. However, as you mention this may not be suited for all the cells. So maybe you can use the same concentration as you selected before, and only remove the cell growth medium to avoid the dilution and the inhibition of fibrinogen conversion into fibrin. Good luck :) Thierry

Hi Thierry and Everyone,

I ended up addressing the problem by reducing my thrombin:fibrinogen concentrations. Previously I was using 0.6 NIH units thrombin per mg of fibrinogen in my sample. Now I use 0.2 NIH units per mg of fibrinogen and perform everything on ice.

I also make sure that my 20 mg/ml fibrinogen stock solution is completely dissolved prior to use, because sometimes precipitates form. (They eventually dissolve if you wait a bit after thawing)

Hi,John,I'm also doing the fibrin gel work,and may I ask how you solve the thrombin with saline, PBS or CaCl2? thank you!@John Yn

Hi Nan Chen , I dissolve the thrombin in DPBS (-Ca - Mg) with 0.1% BSA