View source for C2C12 cell transfection with FuGene

''2 x 6cm dishes with C2C12 cells''

== Materials: ==
{| class="wikitable"
!style="width: 20px" | Day
!style="width: 480px" | Material
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|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: ==
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!style="width: 150px" | Day1:
!style="width: 150px" | Day2:
!style="width: 150px" | Day3:
!style="width: 150px" | Day4:
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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

|
-	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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-	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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-	remove the media and wash the cells twice with PBS
-	add 5 ml of 2% HS-LS to each dish
|}