Help: MIN6 Transfection 96well plate Lipo2000 - (Jan/08/2008 )
Hi, I'm wondering if anyone has successfully transfected MIN6 (P25) in 96 well plate using Lipofectamine 2000 fast-protocol (1 day).
I'm trying to do a high-througput screen. DNA (100ng) is arrayed into each well and all I need to do is add optimem+ LF and then cells, and change the media the next day.
My current protocol is the following:
Hydrate DNA with 10uL optimem
Add in 0.3uL LF2000 in 10uL optimem (after incubating for 5 min)
Add 100uL of media containing 80000 cells
total volume = 120uL
It seems like my cells are clumping to each other before attaching to the plate...
I don't understand why...
I tried without DNA and LF, and it seems like they're still clumping up...
I also tried adding more cells, but it seems like it gets worst...
Any suggestions or protocols?
Andy
It may have something to do with the cell property, which you dont have much can do about. Try to place back the cells soon after plating without delay.
How did you treat your cells before adding them to the wells? Maybe they've been exposed to trypsin too long? (80.000 cells seems quite a lot for one well in a 96-well plate)
After trypsinizing them, I add media/serum to inactivate trypsin... then single cell them, subject them to 3min 300g spin (gentle), discard the supernatant, add fresh supernatant, count the cells that I need, then seed..
MIN6 are very slow growing, and 80000 looks okay.. but I can retry using different amt of cells..
How do you "single cell them"?
Spin down your cells at lower centrifugal force, 100g should be more than plenty.
I usually plate 10.000 cells in 96-well plate (not for transfection, but other purposes) and then my cells are 50-70 confluent the next day already (so, given a doubling time of about 24 hours, I would have about 20.000 cells at that time).
Spin down your cells at lower centrifugal force, 100g should be more than plenty.
I usually plate 10.000 cells in 96-well plate (not for transfection, but other purposes) and then my cells are 50-70 confluent the next day already (so, given a doubling time of about 24 hours, I would have about 20.000 cells at that time).
Just pipette up and down... When you say you plate 10000, is that for min6?
Other cell types. I don't know the size of your cells, but mine are pretty confluent at a density of (estimated!!!) 20.000 cells/well.
And don't pipette up and down too harsh, this causes stress in the cells.
Have you tried plating the cells just in the wells without any other treatment (i.e. absence of DNA, LF2k, even opti-mem)?
15,000-20,000/well seems to be right range for most of the cell lines that I am working with.
we usually have between 20,000 - 25,000 cells/well (different cell lines) in 96 well plate.
Thank you, I will try doing a cell number pilot and see which cell count is the most viable for my transfection.
Has anyone done a MIN6 transfection in 96well with Lipo2000? Thanks..