2 x 6cm dishes with C2C12 cells
Material:
| Day
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Material
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| 1
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2x 6cm dishes
75 cm2 flask
C2C12 cells in a 75 cm2 flask
15 ml FALCON tubes
High serum media for C2C12
TE in PBS
PBS
Neubauer chamber for counting the cells
Pipettes and tips
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| 2
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DMEM (4500mg/l Glucose)
FuGene Transfection reagent
the plasmid which should be transfected
1.5 ml eppendorf tubes
high serum media for C2C12
pipettes and tips
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| 3
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Fresh prepared non-serum-T.i.- media
PBS
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| 4
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PBS
Fresh prepared 2% Horse Serum- low serum
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Procedure:
| Day
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Procedure
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| 1
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- wash the cells with 5 ml PBS
- add 2 ml of TE to the cells and incubate them for 2 min at 37ºC
- during this time: label the new flask and the 2 6cm dishes with the name and the passage number of the cells and the date and fill in the flask 15 ml of new media
- stop the TE with the 5 ml of the HS – media
- transfer the cell suspension into the 15 ml FALCON tube
- centrifuge the cells: 1000xg for 3 min at +4ºC
- remove the old media and dissolve the cell pellet in a few ml of fresh media (the amount of media correlates with the pellet- the cells should be not to dense or to diluted for the counting – general 3 ml are fine for a pellet of one 75cm2 flask of C2C12)
- prepare the Neubauer camber (put the cover slip over the chamber and move it a little bit till you can see the Newton’s rings!!! – this is important for the calculation!!)
- fill between the camber and the cover slip around 40 ml of cell suspension
- count the cells under the scope (all 4 corner crosses - each corner cross has 16 little crosses)- count the both main crosses
- to calculate the number of cells which are in (f.ex. in total 3mls) suspension, calculate at first the average of the 8 crosses, than multiply the average with 104 (the number of cells in 1 ml (!) of suspension) and than multiply this with the amount of ml of the suspension (f.ex. 3) – this is the total number of cells
- pipette in each 6 cm dish 350.000 cells 9next day: use this for transfection) and in the flask 1:30
- 6 cm dishes: fill up to total 5 ml with fresh media
- move the dishes like a cross that the cells are homogenized in the dish
- let them grow at 37ºC, 10% CO2
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| 2
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- FuGene transfection reagent (stored at –20ºC) and plasmid on ice
- For 2 transfections:
o 1) per transfection: 1µg plasmid in 40 µl DMEM
o –for 2 transfections: 2µg plasmid in 80 µl
o DMEM
o pipette this in one 1.5ml eppendorf tube together
2) per transfection: 6µl of FuGene transfection reagent into 154
µl DMEM (total volume: 160µl) into a second 1.5ml eppendorf tube
-for 2.5 transfections: 15 µl FuGene transfection reagent in
385 µl DMEM (in tube one should be now 400µl-> 200µl per
transformation)
"" pipette at first the DMEM into the second tube and
than into the DMEM the FuGene! ""
3) pipette from the second tube (FuGene-DMEM-Mix) 320 µl of the Mix into the first tube (DNA-DMEM-Mix)
4) incubate the FuGene-DNA-DMEM-Mix for 15 min at RT
- during this time: remove the media from the cells and add in each dish 1.8 ml of DMEM- put this for the rest of the incubation time at 37ºC (incubator)
- after the 15 min take the cells put from the incubator and drop wise pipette into each dish 200µl of Fugene-DNA-DMEM-Mix
- incubate this for one hour at 37ºC (incubator)
- after this: ADD 4 ml of high serum media to each dish
- dishes back in the incubator (37ºC, 10%CO2)
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| 3
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- if all wash fine, than the cells should be now confluent
- if this is the case: remove the media, wash the cells twice with PBS and add 5 ml of Non-serum-T.i.-medium to each dish
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| 4
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- remove the media and wash the cells twice with PBS
- add 5 ml of 2% HS-LS to each dish
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