How to seed cell in plates - (Apr/08/2007 )
i always have a problem with seeding 293 HEK cells in 6 or 12 well plate for transient transfection , sometimes i got cell clumps and sometimes not equal distribution, can you help me with this?
To be more specified"
i want to ask about does the centrifugation speed affect the cells? i read here to use may be 100g or so for 5 minutes but my mentor said it doesnt matter about the centrifugation speed as long as your cell condition is good? but i centrifuge cells at 1500rpm and get a hard bellet to deal with?
also i want to ask about pippeting the cells to get a single cell suspension? is it harmful to the cells? cant i vortex gently? how many pippeting times will be ok and not harmful? or does it depend on each cell line?
i am using HEK 293 cells
Have you read this thread: http://www.protocol-online.org/forums/inde...showtopic=21347
i already knew this thread , may be someone can help with the clumping problem i have.
I think longer incubation with trypsin/EDTA may help.
ii also found my 293 cells like clumping , which is very annoying
I get the impression that this is standard behaviour with this cell. They seem to prefer to stay close together rather than spread out over a monolayer. Doesn't mean that they wont spread out though. I don't think centrifuge speed might have anything to do with it. However, i spin at 1000rpm for maybe 2 minutes. Not because its detrimental but because it's not necessary to spin it that long. I can still decant easily without sucking up the pelett.
When I resuspend the pelett with media I pipette it up and down several times to break up the pelett. The media should look like a homogenous cloudy mixture and not with clumps floating around. Seeding them that way could cause them to clump on your culture surface.
To avoid cell clumps i pipette my cells several times up and down after trypsination.
Then i spin them down for 5min 1300rpm.
Had this exact problem myself. HEKs sure like to stick together. The trick is to let them trypsinize. It won't hurt them. Don't rap, or bang the flask to loosen them. Just rock it a little and let the trypsin detach them. When 70 or 80% come off, harvest them. I do it with stable transfected HEKs. I'm sure the surface receptor is chewed-up. But by the next day, never had a problem with the receptor being re-expressed. The experiments are fine. Just be patient! Let the trypsin work. Pipette them a bit, and plate them. Hope this helps. Always works for me.