I am going to detect the M1 gene expression from influenza-infected A549 cells and detection of viral ability using plaque assay from the culture supernatant.
As protocol, A549 cells were infected by virus for 1 hour and then the inoculum were aspirated out. Then, 2 ml of culture medium were added and cultured for 2- 3 days. Finally, the RNA cells were harvested for M1 gene detection and culture Sup. were collected for doing plaque assay.
I'm wondering whether a washing step with PBS should be done prior to addition of culture medium?
Actually, I compared both ways at same time (with/without washing step by PBS). Interestingly, Both of M1 gene expression and number of plaques in group that washed with PBS higher than those of group that without PBS.
Could anyone explain for me?
Thank you!
Is the inoculum toxic to these cells? That might explain higher M1 expression & more plaques with PBS wash.
growth medium usually contains % of serum, washing step is important to it remove all, sometimes it cover the antigenic binding sites needed by all viruses to be able to adsorb and so infect the cells.washing with PBS is important before infection step.
Washing with PBS is important but if PBS damage the cells, it can be washed with culture medium without serum and antibiotics.
Hi,
Yes you should wash with PBS at least 2X and then continue. A549 should not detach with PBS.
Based on standard infection protocols, washing step is important after infection for several reasons including: the removal of the unbound virus, removal of noninfectious virus (virus like particles) that might interfere with the infectious virions in the next round of infection, the inoculum might be a little bit toxic for cell. Also for some viruses, serum concentration might affect the infection step as well. Altogether might reflect why the washing step gave you higher plaques and M1 expression level. anyway I recommend the washing step after infection either with PBS or with media without serum.
Historically, serum interfere with viral infection in vitro. But as Ahmed said, it is not relevant for all viruses. With Human Adv2, for example, I don't see much difference in A549 cells. When you say more plaques, more expression, you need to disclosure more info. Is that statistically relevant? Thant could be a normal variation in your assay too. Specially if you are doing it in different days with viral dilutions prepared in different moments.
You can decrease the amount of serum to 2%. In my hands the plaques are biggers.
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