Hi,
I'm kinda new about handling cells and I'm struggling with contamination lately.
I use HCT116 and they look fine when I culture them in DMEM + 10% FBS + 1% P/S (the first picture).
When I performed transfection experiment, I subcultured cells in Opti-MEM a day before transfection and at this time point I didn't see any implication of contamination and cells looked just fine.
After 4h from siRNA treatment I saw some precipitates so I was kinda confused whether they were bacteria or not but it was just liposomes I guess (they didn't move at all).
And when I checked cells 24h after transfection I saw bacteria. They might not be bacteria, but anyway I was sure they were contaminants because they were moving quite actively in the media (the second picture).
Actually this is the second time I faced bacterial contamination and I already changed Opti-MEM, DMEM and trypsin to fresh ones when I observed the first contamination.
And based on the fact that every time I transfected cells I faced contamination after siRNA treatment, I'm thinking it was just my handling during siRNA treatment or aliqoted siRNA that caused the problem.
Or there might be other possible factors?
I'd really appreciate it if you guys give some advice about this.
I would suggest to use fresh reagents including PBS for washing, Opti-MEM and furthermore before treating with siRNA you are advised to first filter your material (Viral fluid or siRNA containing fluid).
So many times I faced the same problem with bacterial contamination but after adding filtered virus the issue resolved. But in my case i used lantivirus for transfection.
Hope you will get a quality result.
Hello Hansoll Na,
I once had this issue when combining siRNA with Opti-MEM. I did several tests that confirmed that each reagent solely is not causing any death/contamination. However, whenever I was performing the experiments, I had death and contamination. Eventually, the solution was to replace Opti-MEM step with serum and antibiotics-free medium and to date, it works very well.
So briefly, Culture your cells normally and when you do the transfection mix just replace Opti-MEM with serum/Antibiotic free culture medium.
On the next day, refresh the medium of your cells and proceed further with your experiments.
Good Luck
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