Hello everyone!

I am working with H9C2 cell line from ATCC (https://www.lgcstandards-atcc.org/products/all/CRL-1446.aspx?geo_country=se). I cultured the original vial in a T75 flask with the appropriate media sold by ATCC for this specific cell line supplemented with 5% Pen-Strep and 10% FBS. The cells were growing slowly but steadily for 4 days and looked healthy and not contaminated. At day 4 I decided to subculture them into 4 T75 flasks (my usual routine when cells reach 80% confluency). I always add 3 ml of Trypsin/flask after removing media and washing with PBS, incubate 3-5 minutes until detachment and then I collect the cells and add 7 ml of full media to spin at 900 rpm x 5 minutes. After removing the supernatant I resuspend cells in 5 ml full media and use this mix to measure the cells. It was then I saw that 90% of cells were dead! This is the first time I'm working with this cell line, and also the first time this has happened to me using this protocol for subculturing. I took all these 5 ml with another 7 ml of full media and cultured it in a single T75 flasks and they are very slowly growing again, so I'm hoping that they will grow enough for me to try subculturing again.

Any ideas or recommendations on why this happened? I know my incubator is fine and have no mycoplasma contamination, as me and several other colleagues are constantly working with other cell lines simultaneously and they're all fine. So it must be something I did?