Dear all,
I recently performed a proliferation experiment with adult primary human lung fibroblasts (HLF) and observed geometric structures in the medium after TGFbeta treatment (see attached image).
Background information:
- cells: adult primary HLF of a healthy individual, passage 5
- culture medium: DMEM, plus 1% P/S, 0.1 % FBS, Ficoll 70 and 400, Vit C
I am absolutely puzzled and have no idea what those structures might be. Therefore, I am hoping to get your help to figure out what's going on.
Many thanks in advance for your help, it's highly appreciated!
Hey Stephanie!
I think those are ascorbate forming crystals by the addition of Vit C. What is the concentration of ascorbic acid (Vit C) acid ? You can avoid ascorbate crystals by keeping the concentration of ascorbic acid below 50mg/ml. Ascorbate crystals appear when the concentration of ascorbic acid exceeds 50mg/ml.
Hi Raksha,
thanks so much for your reply! I am using 200 µM (~35 mg/ml) ascorbic acid and I also sterile filter the medium before using it (0.2 µm). Do you still think it is due to the ascorbic acid? Do you have any recommendations how to prepare the solutions to circumvent that issue?
Cheers!
Stephanie T. Pohl I agree with Raksha Ganesh that these may be vit C crystals. Just to make sure pour the full filtered medium in a dish, incubate at same conditions for a day and check if they form. If they don't then it must be something in the cell suspension interacting with you medium. If these are freshly thawed cells, and they were cryopreserved in DMSO then it could be crystalizing if you have it at high concentration and haven't diluted the stock properly. Good luck
Hi,
Those are salt crystals. Most probably from ascorbic acid. May be the medium pH could be precipitating (salting out) those crystals. If this happens regularly with DMEM medium, try using different medium. I have seen people using more than 100 mg of ascorbic acid in their assay.
Best,
P. Ramaraj