My 3T3L1-mcherry fibroblast grows perfectly fine, but on differentiation, the cells are getting contaminated. These cells take two weeks to differentiate. For the first week, I grow them in DMEM+10%BCS. For the second week, I follow:
e.g
Monday:IBMX, Dexamethasone, Insulin, and troglitazone-- in DMEM+10%FBS
Wednesday: Insulin in DMEM+10%FBS
Friday: DMEM+10%FBS
The cells are differentiated completely by Tuesday of the third week.
I have been encountering contamination during the second week mostly by Friday; OR the cells get contaminated on Tuesday of the third week, by which it should be ready.
This never happened before by started happening for a month and m unable to figure out why! I also tried making new media, but it's not helpful.
Hi Rachana L. Patnayak . It's a common problem in cell culture and can be due to numerous reasons. Here are a few suggestions/ Sterile Technique: Ensure that you are following aseptic techniques at all times when handling your cells. This includes sterilizing all work surfaces, pipettes, and other tools before and after use, and handling cells in a clean and regularly disinfected biosafety cabinet/ Media and Reagents: Although you've already tried making new media, consider checking all other reagents (including PBS, trypsin, and any other solutions you use) for contamination. Also, ensure that the FBS and BCS are not expired and have been stored properly./ Water Baths: If you're using a water bath to warm media or other solutions, make sure it is clean. Water baths can be a source of contamination if not regularly cleaned and disinfected./ Cell Storage: Check your incubator and ensure it is clean and at the correct temperature and CO2 level. Regularly disinfect the incubator to reduce the risk of contamination/ Cell Culture Flasks and Plates: If you're using reusable glassware, make sure it's sterilized properly before use. If you're using disposable plasticware, ensure they are from a reliable source and are not expired./ Media Change Schedule: It could be possible that your cells are undergoing stress due to the frequency of media changes and the differentiation process, which might be making them more susceptible to contamination. You could try changing media more frequently to see if it helps /Myoplast Contamination: In rare cases, myoplasts can be a source of contamination in differentiated 3T3-L1 cells. These are small, round cells that arise during the differentiation process. They can overgrow and contaminate the adipocyte culture.
Try filtering through 0.22um your differentiation reagents. This multicomponent mixture seems to bethe most probable source of contamination.
Hello Rachana L. Patnayak
You are not facing any contamination during the culture of the cells. You are facing contamination after the differentiation. All the materials which you are using are the same except the differentiation cocktail.
Specifically, your insulin might be contaminated in the differentiation cocktail but again you need to confirm. if you want to test, just seed the cells with alone Insulin, IBMX, Dexamethasone, and TZD, or in some combination and check the contamination. But it'll take your time.
So As Anatoliy Zubritskiy suggested try to filter the differentiation cocktail from a 0.2-micron filter.
If you want, you can prepare fresh differentiation cocktails and use them after filtration.
and take care of everything that Saman Behboodi Tanourlouee suggested.
Thank You
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