MCF7... dead.. - (Jun/15/2006 )
I seeded MCF7 on 96 well plates yesterday and subcultured the rest at the same time to T25 flasks. Today I found that all my MCF7 cells are floating, and shrunk, which pretty much means they're dead, with very few cells attached to it. The cells that are in the 96 wells looks okay though.
Anybody have any idea of why this happens? I doubt it's contamination of any sort.
Anybody have any idea of why this happens? I doubt it's contamination of any sort.
May be the MCF7 was trypsinized for too long before putting them in the T25 flasks.
was the media in the T25 the same as the 96 wells?
was it a new flask, or did you reuse an old flask? really shouldn't make a difference, but, still.
did they look sick before you started playing with them?
do any other cells (belonging to you, or other people) look sick, or have they died?
you'd would have had to trypsinize them for a least a 1/2 hour for them to have died from that.
vetticus
Trypsinized too long, very unlikely. It was all neutralized with medium immediately before splitting for subculture and seeding.
The media was from the same bottle, definitely, but I added extra fresh media into the T25 flask.
And reusing old flask has been working pretty well this past few weeks, so that can't be the problem.
They look pretty happy before I start playing with them.
The MCF7 that my colleage cultured in T75 flask and sat right next by my t25 flasks look very happy indeed.
I am thinking that it's gotta be some steps that I subconsciously did differently than any other day.
Anyways, I am trying to revive them by flushing all the dead cells out and see how much I can salvage by adding fresh media. Will let you all know if it is beyond salvation.
The cells are now resting in peace...
Apparently it is beyond salvation. Oh well, good thing got another flask going.
Apparently it is beyond salvation. Oh well, good thing got another flask going.
It is hard to guess exactly what had happened. Here are some possibilities to think about: 1) you might forget to lose up the lid a bit, so no CO2 gets in and pH is too high, if that is the case the medium color will be purple wish than usual . 2) by any reason that you might have used T25 flask designed for suspension culture only and not those surface treated for adherent culture? 3) Any possibility due to contamination ( the medium turn turbid, yellowish, with a lot of small particles) ?
