Hello
Hello
Culture conditions for MEF feeder cells ( DMEM containing 10% FBS, 2 mM glutamine,1x10-4 M nonessential amino acids and 50 U, and 50 μg/ml penicillin and streptomycin )
protocol culture
1)Remove the vial from liquid nitrogen and thaw quickly in a
37°C water bath.
2) Remove the vial from the water bath as soon as the cells
are half-thawed, and sterilize the tube by spraying with
70% ethanol.
3) Transfer the cells with 10 ml of MEF medium to a 15 cm
conical tube and pellet the cells by centrifugation at 200 g
for 5 min.
4) Discard the supernatant and resuspend the cells with 10
ml fresh MEF medium and plate the cells at seed density
of 104 cells/ cm2.
5) Incubate at 37°C with 5% CO2, until the cells reach 80-
90% confluency.
6) Change medium twice a week or when pH decreases.
4. Passaging MEF cells
Cells should be split when they reach confluency. We recommend
splitting the cells based on 0.5x104 cells/ cm2.
1) Discard the medium and wash the cells twice with PBS.
2) Aspirate PBS, and add 1 ml per T75 flask of 0.25%
trypsin-EDTA, and incubate for 1 min.
3) Add 5 ml of MEF medium, and break up the cell clumps by
gently pipetting up and down several times.
4) Transfer cells into a conical tube and centrifuge at 200 g
for 5 min.
pleas , read the attachment , i hope help you
Good luck
Dear Bhairavi,
Please see the following link.
http://www.systembio.com/downloads/Manual_iPSCellLines.pdf
It may help you.
Please refer to the following link:
https://www.lifetechnologies.com/jp/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/culture-ipscs-conditioned-medium.html
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