C2C12 cell transfection with FuGene

2 x 6cm dishes with C2C12 cells

Material:

Day	Material
1	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
2	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
3	Fresh prepared non-serum-T.i.- media PBS
4	PBS Fresh prepared 2% Horse Serum- low serum

Procedure:

Day	Procedure
1	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
2	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)
3	- 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
4	- remove the media and wash the cells twice with PBS - add 5 ml of 2% HS-LS to each dish