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Trouble with making Collagen-Matrigel 3D Cultures

Collagen Matrigel 3D Culture breast cancer cells

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#1 catherine_mae03

catherine_mae03

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Posted 21 September 2012 - 06:00 AM

Hi,

I am having problems setting up my Collagen/Matrigel 3D culture. I have been following a protocol close to this one that I have linked here...http://muthuswamylab...ls/Invasion.pdf

We use Collagen 1 derived from Rat Tail from Sigma (concentration 4.2 mg/ml), which we borrowed a small aliquot from a different lab - so I never prepared this collagen solution. We are mixing in 10XPBS and 0.1M NaOH. We don't need to add any HCl because the concentration of the Collagen mixed with the PBS and NaOH is 7.4. Next, I mix it 1:1 with Matrigel on ice. Then I plate this mixture in a 12-well plate. I let the matrix solidify for 3 h at 37 degrees C. Then when I go to plate my cells that are in a 2% Matrigel 3D culture medium solution, I pipette very slowly down the side of the well, but what happens is that the Collagen/Matrigel matrix that I plated on the bottom of the plate seems to like melt away with the addition of the cells. It doesn't stay solid like when I plate Matrigel alone.

Has anyone ever had this problem or know what I might be doing wrong?

Thank you,
Cat





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