Dear readers, I am a master degree student currently working on phototoxicity assessment using Balb/c 3T3 cell line. However, I have encountered some technical issue during the running of this assay, for both UV- and UV+ exposed well plates. The cells (passage number 20, cultured in DMEM + 10%Calf serum +1% Pen-Strep 5% CO2, 37°C ) get rounded and detach after the first and in particular after the second washing step required by the test guideline at day 2 after the 1h and 50 min incubation with the test substance (1h in incubator and 50min in dark or under UV-light exposure). I would underline that, in my case, the test substance consist of only 1% DMSO in HBSS (Mg+Ca+) because the issue of detachment occurs even with this vehicle (we have tried also with just HBSS but we obtained the same issue).
Do you have any clue on how to troubleshoot this problem? Thank you in advance.
Material:
- 96-well plate NUNC 96-well plate (#167 008) (I have tried also with poly-lysinated or collagenated plates, both were unsuccessful)
- Buffer for washing: warmed HBSS (Mg+ Ca+)
- 8-pin manifold to remove buffer (we have tried also to invert the plate over a absorbent paper, but was unsuccessful)
- Serum used: Calf serum (we have tried also with FBS or with combination of Calf+FBS, both were unsuccessful).
Check https://www.oecd-ilibrary.org/test-no-432-in-vitro-3t3-nru-phototoxicity-test_5lmnfnj6bkq5.pdf
Hello Luca,
You can check on this paper for help.
https://www.scielo.br/j/abd/a/fcdGxp6q3g9xDMcxdd6rJnv/?lang=en
Luca Romito Hi Luca, Did you manage to solve this issue at all please? I am experiencing the same exact issue and would welcome any advice. Many thanks.
Dear Dr. Jodie Ball, we have successfully solved the issues. Please check my thesis published on my profile in the "Research items" section, hoping it is helpful for your project. For any other information you can send me a private message.
Dear Luca Romito , Thank you so much for getting in touch and offering help. I will be sure to check your thesis :) Thank you very much once again, Jodie.
Dear Luca Romito, thanks for sharing. I wish I saw it earlier. Unfortunately I had to go myself through all the same optimizations steps of the assay before I came across your thesis. Same conclusions although. So, I do recommend all who is going to apply the method to read Luca's thesis first.
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