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.
