Protocol for transfection of H4 , ( ATCC® : HTB - 148 ) Cells by FuGENE HD in 96 well plates .
Cell plating H4 ( neuroglioma ) cells were seeded from 90 - 100 % confluent culture the day before transfection with the density 7,000 cells / well in 100μl complete growth medium ( DMEM + 10 % Fetal Bovine Serum ) .
For transfection in 100 % Fetal Bovine Serum the complete growth medium was replaced with Fetal Bovine Serum two hours before transfection .
Complex preparation ( per 20 wells ) .
Tissue culture 96 - round bottom well plates were used for complex preparation : Prepare 0.02μg / μl pCMVβ DNA solution in OptiMEM® or sterile deionized water .
Add 6 μl of reagent to 100 μl of DNA solution .
Mix carefully by pipetting ( 15 times ) .
Incubate 10 min at room temperature .
Add 5 μl of complex per well to the cells , and mix thoroughly .
Incubation .
Incubate transfected cells in CO2 incubator for 48 hours .
Detection of β - gal expression .
Remove the medium from the well and wash the cells once with 100μl per well PBS .
Fix the cells in the well with 50μl solution of 4 % formaldehyde in PBS for 5min at room temperature .
Wash each well twice with 100μl PBS .
Add 50μl per well of substrate / stain solution and incubate the plate overnight at 37°C .
Observe the cells under microscope and evaluate the proportion of blue ( β - gal - positive ) cells .
