I have THP1 culture but it was contaminated. I have used pen-strep (Sigma) and also tetracycline antibiotic solution but still it didn't kill the contaminant (Brownian movement). Can anyone offer advice? I am using T25 flask for THP1 culturing.
maybe your stock is contaminated, try defrosting a new stock
I culture THP1 cells using RPMI Medium 1640 (Gibco) containing 10% heat-inactivated FCS [Gibco-BRL], 1% 1M Hepes solution pH 7.4, and 1% penicillin-streptomycin, in 25-cm2 flasks. The cultures are keep at 37°C in 5% CO2 at 95% humidity, and I change the medium every 2 days. But when I defrost the cells, I culture the cells with 20% FCS for a few days.
Hi Senthil. I agree with Loreto, maybe your stock is contaminated and you should first consider that. Another thing that you may want to do is to decontaminate the CO2 incubator. Autoclave everything and inclusive the water you put on the tray. I usually use copper sulfate in the water and change it every week. Check the water bath that you use to warm up the media. In general, these are the problematic itens! Hope that helps, all the luck. Best.
I would suggest the following troubleshooting procedures:
firstly put some media you are using in a petri dish and keep it in the incubator to check whether there is anything wrong with the media.
after that check your incubator properly whether its clean or its developing any spores.
then lastly try with a new vial if all these things are okay.
agreed with Loreto. Also keep the flask vertically. Check after every two days.
Firstly you must be sure your equipment (media, tyrpsin, serum, LAF, CO2 incubator and lab conditions). I prefer serum with gama irriadiated. If you sure all of them you should check your stock cell. You can harvest stock cells to bacteriologic agars (TSB and FTM). In addition you can different cell line in your conditions. So that you can check easily your conditions.
Hi Senthil. I agree with the others. If you follow these tips you should be free of contamination easily. When we have contamination episodes, we discard the contaminated cells and the media aliquots that were in use and decontaminate the incubator. After that, we defrost a new stock. This means, a fresh start. Related with the growing media for THP1 cells, we use the same media as Loreto explained, but we add 2-mercaptoethanol to a final concentration of 0.05 mM (this is suggested by ATCC). This helped a lot to our THP1 cells.
I agree with Loreto and Tarun, your cells are contaminated and you should looking for the source of contamination: put the medium you are using in a small petri dish and keep it in the incubator for 48h to check whether it is contaminated. As already suggested you should de-contaminated all your cell culture staff.
Check also whether you have a Mycoplasm contamination, using a commercial kit. In the case of Mycoplasm contamination the cells grow very slowly and you can cure them using some reagents like Plasmocin .
I suggest use of gentamicin which is much more effective against contamination.
I agree with previous suggestions. you need to defrost a new stock !!!
Than I suggest you take the following precautions
Keep all work surfaces free of clutter.
Only handle one cell line at a time. This common-sense point will reduce the possibility of cross contamination by mislabeling etc. It will also reduce the spread of bacteria and mycoplasma by the generation of aerosols across numerous opened media bottles and flasks in the cabinet.
Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between operations and allow a minimum of 15 minutes between handling different cell lines.
Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced at regular intervals.
Don't have too many people in the lab at any one time.
Avoid keeping cell lines continually in culture without returning to frozen stock.
Avoid water baths from becoming dirty by using Sigma Clean
I think it's better to defrost a new stock.
It's almost impossible to delete completely the contamination from the flask with cells
One question:
the culture medium is clear or not? if it is clear probably you have a contamination by mycoplasma!! wash the incubator and defroze a new stock.
I also agree with Loreto, and culture my cells almost exactly the same way that she does. However, when I was looking into the ATCC guidelines for THP-1 culture, it recommends 0.05 mM beta-mercaptoethanol - I tried this starting a couple days after bringing the cells out of liquid nitrogen and it seemed to work well.
You may got that contamination due to your handling problem. Wash the cells with PBS and Pen strep twicely. Add gentamycin and Penstrep. But, it cause the suppression of cell growth. If you recruit your leukemia cells from contamination. then you can get your cells as healthy by using 15-20% FBS and conditioned media.
MY DEAR RESEARCH FRIEND , Thank you very much for your valuable advice.
Hi,
I will start culture THP-1 cell line and I am wondering if it is possible to replace beta-mercaptoethanol with DTT in the culture medium?
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