Measurement Standard

08/08/2010

seeding cells for test measurements - 96 well plate for plate-reader and FACS

cells were grown in DMEM 10%FBS with phenol red
washed with PBS
trypsinised (2 ml trypsin)
5ml of OptiMEM media (no FBS) added
counted cells: HeLa 3.8*10^6 cells/ml, HEK 3.3*10^6 cells/ml, HEK T-Rex 1.5*10^6 cells/ml, Huh7 0.8*10^6 cells/ml
seeded 5000 and 2500 cells/well as on the scheme 96well plate 080810.jpg
media: DMEM +L-Glu +PenStrep +10%FBS, OptiMEM +PenStrep, OptiMEM +PenStrep +2%FBS
in all wells where T-Rex cells were seeded zeocin and blasticin were added, in wells with Huh7 cells - non essential amino acids

-> cells did not adhere well, coat plate with poly-L-lysine and repeat

seeding and transfecting cells for microscopy test measurements

add 0.6ul FuGENE reagent to 20ul of OptiMEM
mix and incubate 5' at RT
add 0.2ug DNA
mix and incubate 15' at RT
add DNA-FuGENE solution to 10 000 cells (400ul)
mix and incubate 10' 37degC 300rpm (shaker for 15ml falcon tubes is in the cell culture room at 3rd floor)
transfere cells to the plate
grow 48h

HeLa, EGPF	HeLa, EBFP2	HeLa, EGPF and EBFP2	HeLa, EGPF and EBFP2 1:10
HEK T-REx, EGFP	HEK T-REx, EBFP2	HEK T-REx, EGPF and EBFP2	HEK T-REx, EGPF and EBFP2 1:10

coating plates with poly-L-lysine

add 15ul of poly-L-lysine solution to each well (make sure whole surface is covered with solution)
leave for 30' in the incubator
remove poly-L-lysine solution
wash once with PBS

new 96-well plate was prepared (wells 2500 cells Huh7 are contaminated with HeLa) like previously

seeding cells for pilot FACS and Tecan measurements (96-well plate)

transfection - 96-well plate

pilot FACS and Tecan measurements (96-well plate)