Hello, I am new to working with HEK-293 cells and am having some issues. I was told that as they are loosely adherent in the T75 flasks that I use in order to split them all i had to do was aspirate the media, run fresh media down the plate several times to remove the cells then split accordingly. I do this and the next day the cells have completely clumped together, so much so that you can even visibly see it in the media. I am using 10% FBS DMEM with 1% Pen strep. I am expressing a membrane protein so I was told i can't use trypsinaisation. Any suggestions on how I can prevent this from happening? thank you
Obviously the membrane protein could change things but when we passage HEK293 cells, we either pipet media up and down to dislodge or simply tap the flask a few quick times. This dislodges them but not in single cells, in large clumps and sheets. Next you need to take a serological pipet and pipet the cell suspension up and down. You can be fairly vigorous with it, trying to break up the clumps into a more single cell suspension. Then go about your replating. Also make sure with these cells that you don't split too thin. We have found that while they grow quite fast, they do not like to be too thin. They are a cell line that relies on close proximity to other cells so I don't split more than 1:12 or so.
We work with a membrane channel protein and we use trypsin, only a couple of minutes and then we dilute with DMEM, centrifuge and discard the DMEM with trypsin. Then, we add fresh medium and split HEK293 cells. We had not had any problem with this. And also, before trypsinaisation, we aspire the medium and wash with PBS 1X.
HEK293 can easily be detached by washing out with growth medium. There is no need to trypsinize. Just take the 10 ml pipet and wash them out by pipeting up and down. Place in a 15 ml tube and centrifuge 3 min, 800-1000 rpm. Aspirate the supernatant and first resuspend the pellet in 200 ul of growth medium. Gently resupending cells in a small volume (pipet tip of 200 ul) will brake cells clumps. Since you don't use Trypsin-EDTA you will have many clumps (EDTA helps braking the clumps). Once you resuspend your cells well, you can add the rest of the medium and make you dilutions for subcultivation. Suculture ratio should be 1/10.
Hi Nichole Owen and Milica Bozic !
Do you have any paper or protocol reference for HEK293 cells detachment with just pipetting (not using trypsin or chemical method).
Please if you could share it.
Hi Kaneez Fatima
I do not have any paper or protocol reference.
However, maybe you could find some references on the ATCC web page: https://www.atcc.org/search#q=HEK293&sort=relevancy&numberOfResults=12
Best,
Milica
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays