b)
Plasmidic preparation of the ADN
The ADN plasmidic is obtained by culture of bacteria
transformed beforehand by the plasmides of interest, in medium LB plus
ampicilline 1X. The ADN plasmidic is prepared according to the protocol of
plasmidic purification (Plasmid Purification, Qiagen). It undergoes then a
precipitation with cesium chloride, this stage increases the purity of the ADN
but is not obligatory. The quality of this ADN is analyzed by spectrophotometry
to evaluate the quantity and migration on freezing of agarose 1% to evaluate
quality.
c)
Transfections cellular
1) Transitory Transfection of Cos-6 cells by calcium
phosphate
Twenty four hours before the transfection, the cells are put
in wells of culture 6 cm in diameter at a concentration of 600000 in 5 ml of
medium. The next day, the medium is replaced by 4 ml of fresh medium. A
solution of 1 ml containing 5 to 30 ug ADN plasmidic in 450 ul of H2 O, 50 ul
of CaCl2 (2,5 M) and 500 ul of HBS 2X (NaCl 280 mm, KCl 10 mm, Na2HPO4 12 mm,
Glucose 12 mm, Hépès 50 mm) beforehand incubated 30 min. at
ambient temperature is added on the cells. They are given in the drying oven 12
hours, then washed with the PBS 1X. Fresh medium is added and as well as the
ligand, possibly. The cells are again incubated 12 midnight for a proportioning
of the luciférase at 2 days for a proteinic extraction with 37°C.
Before the extraction of proteins, the cells are washed with the PBS 1X.
2) Transitory Transfection of cells HL-60 by
electroporation
The cells passed the day before to the usual concentration
corresponding to the cellular type and the tanks with electroporation are
cooled beforehand with 4°C. Each tank is used for 30x106
cells. Three hours before the transfection, the cells are put at
1x106 per ml then incubated with 37°C. Then they are
centrifuged, washed and gathered with 4°C in a volume of
OPTIMEM-1 equivalent to 10 ml for 100x106 cells. They are then
included in a volume containing 100 ul OPTIMEM-1 by tank. After
placehaving placed 100 ul in each tank of them, the mixture is incubated during
10 min. with 4°C. The cells are added in the tanks, with a
plasmidic solution containing 100 ul OPTIMEM-1 by tank. The
electroporation is carried out in a electroporator of which
parameters are: C= 960 uF and V=250 V, they are then put
to incubate at ambient temperature during 30min. The cells are
recovered with 1 ml of complete medium heated beforehand with 37°C.
The cells are incubated with 37°C during three hours, then given
in culture.
3) Transitory Transfection of
K562 cells by the lipofectamine
The ADN plasmidic is prepared in a volume of 100 ul per well
with 5% of lipofectamine more and supplemented by 95 ul of OPTIMEM-1 which is
left incubated during 15 min. a solution of 12,5 ul of lipofectamine and of
87,5 ul of OPTIMEM-1 by well is prepared. The two solutions are mixed
in sterile polypropylene tubes, by adding the plasmidic solution drop by drop.
This mixture is incubated one hour at ambient temperature. The
cells are conditioned with the number of 2x106 per well in 0,8 ml of
OPTIMEM-1 (the concentration initial of the cells in their medium was 500000
per ml) and incubated one hour with 37°C. The 200 ul prepared are
deposited drop by drop in each well. Then the cells are incubated two
hours with 37°C and finally 3,5 ml of medium are added and the ligand
éventuellement.d
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