I have been experiencing issues with my HL60 cell cultures. They always seem to get infected eg. 10 days after being in the culture. All my other cell lines seem to be doing fine and I checked all of my reagents and they are contamination- free I also change reagents and use new ones or supplement the cultures with extra pen/strep but I still get infections. Also, what is more weird is that every single time I seed them in a 96 well plate they get infected 2 days post seeding even though my initial cultures are not infected.
Does anyone else have problems with this cell line? Do they get infected easily? Do I need to change my antibiotic? I am confused.
I have cultured the cell line for about 2 months. I didn'nt find that problem, but I did with k562. You can seed less in the flask to check whether they are infected.
Are you sure that all of your pipettes/pipette tips and other equipment you are using are completely sterile? In theory you should be able to do TC without using any antibiotics if your technique is good (though obviously it is easier to supplement with antibiotics). Are you 100% sure that it is not the starting culture that is infected? It may be that the infection is there but you can only see it after so many passage numbers. The culture may have been contaminated upon freezing for storage. Is the cell line from a regulated source e.g. ECACC/ATCC? Sorry for all the questions but there are clearly many possibilities as to why it may be becoming infected which need to be negated.
Hi Konstantia my advice is to change this line and replace by new HL60 cell line as this line is my be contaminated by mycoplasma to sure that take loopful from the batch and make sterility test before culturing
regards
The reason could be that your initial HL60 culture was contaminated somewhere along the way. If you haven't obtained it from a reliable source, then it could've been that the culture you obtained was lightly contaminated. Alternatively, your culture could've been contaminated and then frozen and now everytime you thaw it, the contamination reappears after some time in culture. Of course, there is a possibility that your culture medium or your labware is contaminated but that should be something specific to your HL60 cells since you don't report any contamination in your other cell lines. Is there anything specific for the HL60 cells? Something that you use exclusively for these cells? You mentioned 96-well plates; have you seeded the other cell lines in these plates? It could be that they are non-sterile or their sterility has been compromised. As mentioned earlier, the use of antibiotics in cell culture is not obligatory; I have cultured cells for years without the use of antibiotics or antimycotics and I never had any issues with contamination. It will take some time and effort to isolate the cause so good luck!
I also have encountered problems similar to yours, the best advice i can give you based on my experience is as follows:
Does your CO2 incubator have a water pan?- this is a major source of fungus. Check the pan is grime-free, change the water every 2-3 weeks and add a spoonful of copper(II)sulphate to the water per liter. Otherwise check inside your incubator-make sure you can clean EVERY SINGLE INCH of the inside with a disinfectant. Dismantle the trays and inner back covers (that cover the fan) whilst cleaning too- you'll be surprised what you may find at the back! Its time-consuming BUT essential.
Its also very possible that your flasks, 96well plates and tips are contaminated or kept in a non-sterile area. Use only those that are individually sealed and tips that are autoclaved.
Overdosing your cultures with antibiotics is a bad idea- as this will surely make them resistant at some point. So try avoid this. Im sure that the cause is your tips or plates
Thank you everyone for your reply!
Actually I have been experiencing this problem ONLY with this specific cell line and I have been using same tips and pippetes.
Only thing I can think about is that this cell line was contaminated when I recieved it from a coworker.
Is there any posibility that my lentivirus causes some sort of infection on the cells I transduce?
There is a very real possibility that the cell line could have been contaminated by someone else if you received it from another person. The lentivirus should not cause any other infections other than the lentivirus itself. However, if the preparation of the virus was not done correctly under sterile conditions then yes this is a possibility. However, are you also seeing infection in the parental line or just the transduced lines?
It was both the parental cell line and the transduced one.
However I threw the infected HL60 cells away and then thawed some that I had frozen when I first recieved the cell line and they dont get full of bacteria. I notice some circular modules between the cells that might be moving by themselves, yet they arent many and they dont move the way bacteria do in a cell culture. I also wash the cells using lower rpms , lets see if that proves helpful.
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