Matrigel invasion assays. Need a fixing solution - (Jun/23/2007 )
Wonder if anyone is doing or has done invasion assays using the pre-coated matrigel transwell-inserts.
The problem i am having is that lots of my cells come off from the bottom of my membrane after i try to fix it using the DIFF quick fixing solution that contains methanol. I can actually see dead cells floating in my container holding the fixing solution
Wondering if anyone has used other methods of fixing and staining the cells attached to the bottom side of the membrane.
I had an idea of cutting out the bottom membrane of the transwell inserts, placing it upright on a glass slide, fix cells using methanol, stain with Hoechst, and count using immunoflurescent microscope. Any opinions?
The problem i am having is that lots of my cells come off from the bottom of my membrane after i try to fix it using the DIFF quick fixing solution that contains methanol. I can actually see dead cells floating in my container holding the fixing solution
Wondering if anyone has used other methods of fixing and staining the cells attached to the bottom side of the membrane.
I had an idea of cutting out the bottom membrane of the transwell inserts, placing it upright on a glass slide, fix cells using methanol, stain with Hoechst, and count using immunoflurescent microscope. Any opinions?
It sounds strange. What about procedure of your staining. After invasion I add 1 ml fix solution into 24 well plate and place insert in the plate carefully. After incubation period I remove fix solution, wash two times and place insert in dye, previously added into the plate. It was no problem. Usually I use Asur-Eosin staining ( according Romanovsky method). But with diff-quick it works well too.
Thanks for the reply circle point,
I use about 750ul of the fixing solution in 24 well plate and i leave the transwell in the solution for 4 mins. But i use the same fixing solution for multiple transwells (maximum 12). Do u think that has to do with something?
Also, what do you think about skipping the whole fixing thing and just cutting the membrane out, putting it on glass slide, fixing with methanol and then staining with Hoechst. I can then count the cells under the microscope.
I am interested to know whats in your fixing solution?
I know that some people use 1% paraformaldehyde in PBS to fix cells in 3D matrigel cultures...
D