View source for A1 cell transfection with FuGene

== Materials == 
* Fugene Transfection solution
* Complete media for A1 cells
* No serum media
* ATE
* APBS
* 10 cm dishes
* eppendorf tubes

== Procedure ==
# Incubation FuGene – DNA:
# 18 µl FuGene : 6 µg DNA if the transfection has to be done in 10 
cm dish. Downscaling or upscaling for different dish sizes are possible (ex. 6 µl for 6 well plates)
		NOTE: if the transfection is a cotransfection use amounts of 
each plasmid so that the sum of all the DNA in the solution is 
6 µg
# The Protocol is the same suggested from the manufacturer: prepare DNA in the appropriate number tubes in A1 media without serum (200 µl/ tube). In parallel prepare a master mix of FuGene in no serum A1 medium (200 µl each transfection), incubate the mix 5 min @ RT than add the FuGene containing medium to the DNA containing tubes.
# Incubate 20 min @ RT.

	Cell preparation:

		In the meantime prepare the cells: for one 10 cm dish 
transfection we need one 175 cm flask, detach and seed into a 
10 cm dish, following the same protocol we used for passage A1 
cells.
Briefly:
-	Wash the cells with 5 ml APBS
-	Detach them wit ATE and stop the reaction with 5 ml HS – media 
-	Pellet them by centrifuging them for 3 min @ 1000 rpm
-	Resuspend in complete media
-	Seed the cells in 10 cm dishes

Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously in the in the plate

Incubate the cells @ 25 degree 2% CO2 (after 2 days check)