C2C12 cell transfection with FuGene

2 x 6cm dishes with C2C12 cells

Materials

Day	Material
first	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
second	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
third	Fresh prepared non-serum-T.i.- media PBS
fourth	PBS Fresh prepared 2% Horse Serum- low serum

Procedure

Day	Procedure
first	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
second	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)
third	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
fourth	remove the media and wash the cells twice with PBS add 5 ml of 2% HS-LS to each dish