I perform Spheroid assay for 3D spheroid by Hanging drop technique. I am using matrix of collagen from rat tails and therefore my matrix is not clear. I think it is participate of collagen as it is white fibrous whan see in well and debris in microscope. From this problem the impact for spheroid is not good, not circle, cell of this expose form there in next time so don't see abnormal shape in first day. I think this problem happens as collagen hasn't well of polymerization. How can do it? I put collagen on ice 3-5 minitue.
ัyes, I used matrigel mixing with cell suspenion . Then I mixing there with collagen (matrix =collagen stock solution +10XDMEM+NaOH ) So drop on well plate. Incuate in CO2 30 minute and added media.
On 1 st day Spheroid are round It's ok. On 2 nd cell remove on spheroid or not round. I saw collagen not clear it precipitate. I read some paper added NaHCO3 in collagen but in my protocol don''t added it.
What is your opinion?
Thanks you so much .^_^
That 's good for your answer.I will try to do it and use pH indicator for scale color.
Hi Suwadee,
Did you solve your problem about collagen gel polymerization? Mostly, this problem is related to the pH change of the solution mix. In my collagen solution I add MEM medium and NaHCO3.
NaHCO3 is also important for it, you can see the colour chaging in your polymer solution if pH is not correct. it means if you have NaHCO3 you will be sure if the solution ok or not?
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