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Problems with C2C12 resuscitation

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Problems with C2C12 resuscitation

Postby CecileL » Wed Jun 23, 2010 7:50 am

Hello everyone.

This is my first post here, and I hope you could help me.
I'm restarting a lab, and I'm experiencing trouble thawing my C2C12 cells (mouse myoblasts).

I thawed my ampule rapidly in a 37°C waterbath, and wipe my ampule with ethanol.
I then pipette the content of the ampule in 5 ml of prewarmed media (composition below), centrifuge that suspension at 1100 rcf / 5 minutes, resuspend the cells in my media and plated my cells. (Before centrifugation, I processed a Trypan Blue stain. All the cells were viable.)
I plated the cells at a density of 22 000 cells/cm², and incubate at 37°C 5% CO2.

That was yesterday in the early afternoon.
Today morning, a lot of cells haven't attached and are floating, and the cells that adhered don't look very well.

Maybe it's too soon to worry myself, but I tried to resuscitate another ampule last week and all the cells died (my density was way too low!!!).

Do you see anything wrong in the way I thawed my cells?
Do you know the C2C12 cells, and/or do you have protocols to give me?

Here is my medium composition:

DMEM: Sigma (D5671) 500mL
Pyruvate Na: Gibco (11360-039) 5mL
NEAA: Gibco (11140-035) 5mL
Pen Strep: Hyclone Thermo (SV30010) 1mL
Fungizone: Gibco (15290-018) 0,5mL
FCS: Sigma (F2442) 50mL
L-Glutamine: Sigma (G7513) 5mL

I really hope someone could help me.
Thank you very much in advance for your help.

Cécile
CecileL
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Postby CecileL » Wed Jun 23, 2010 12:10 pm

I put a flask with the same amount of medium than in my culture, and place it in my incubator. The medium turned to pink in 2 hours whereas there's no cells in it!

Can it be due to an excess of CO2? Or a lack of CO2?
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