Hi,
I am introducing the strategy briefly which I follow to infect the MC38 cells with harvested lentivirus media from HEK293T cells at 24 and 48h.
1. First, MC38 cells are cultured in 60mm culture dish and when cells growth reach at 70% confluent, I change the previous media to virus medium for transduction with the help of polybrene (8ug/ml).
2. After that when cells growth is completed, I subculture the first-time infected cells and again transduction is done with viral media for second time when cells volume is 50-70%. Then the dishes are kept on incubator for overnight or more based on cells growth.
3. Finally, GFP positive cell percentages are determined by flow cytometry after proper growth. The problem is that GFP% is very low (
Have you checked mycoplasma contamination? I've grown cells that were positive, removing the infection boosted our GFP/transfection rate significantly. They are a known source of transfection inhibition and are more common than thought. They do not behave like fungal/bacterial contamination either (being more subtle).
Do you spin down the plate with the virus media and polybrene on it before incubating? I usually do 1000 rpm for 30 minutes.
Can Kiessling, thank you so much for your kind response. I didn't check the mycoplasma contamination. Sometimes my cells are infected with some bacteria, and I wash my cells with a 10% PS solution in culture media to remove that.
Ermela Paparisto, I don't spin down the virus plate. I just collect it by using the filter membrane and then directly add it to the cell culture plate with polybrene.
PS will not work against mycoplasma, you need antimycotics. You will not notice mycoplasma contamination with phase contrast microscopy or just by looking at the culture media. You need to do a test to detect it because it is so subtle (like the one here https://bioscience.lonza.com/lonza_bs/CH/en/Cell-analysis/p/000000000000186472/MycoAlert-Mycoplasma-Detection-Kit-%28100-Tests%29)
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