X-Gal staining of Hela cells 72 hours after transfection by FuGENE®HD and Optifect® at various conditions

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FuGENE®HD	Optifect®
Reagent / DNA Ratio 0.3μl/0.1μg 5μl Complex per well Complex Incubation 5 min	Reagent / DNA Ratio 1.5μl/0.25μg 5μl Complex per well Complex Incubation 5 min
FuGENE®HD	Optifect®
Reagent / DNA Ratio 0.2μl/0.1μg 5μl Complex per well Complex Incubation 15 min	Reagent / DNA Ratio 1μl/0.25μg 5μl Complex per well Complex Incubation 15 min
Transfection with competitor reagent was performed according to manufacturer’s instructions

Protocol for Transfection of Hela Cells, (ATCC®: CCL-2.2™, Human cervix adenocarcinoma) by FuGENE HD in 96 well plates.

1. Cell plating.

   HeLa cells were seeded from 90-100% confluent culture the day before transfection with the density 10,000 cells per well (optional cell density as low as 1,000 and 3,000 cells per well can be used) in 100 μl complete growth medium DMEM + 10% Fetal Bovine Serum.

2. Complex preparation (per 20 wells).

   Tissue culture 96-round bottom well plates were used for complex preparation:

   1. Prepare 0.02μg/μl pCMVβ DNA solution in OptiMEM®.
   2. Add 6 μl of reagent to 100 μl of OptiMEM® / DNA solution.
   3. Mix carefully by pipetting (15 times).
   4. Incubate 10 min at room temperature.
   5. Add 5 μl of complex per well to the cells, and mix thoroughly.

   Optimal ratio of reagent/DNA varies from 0.25μl/0.1μg to 0.4μl/0.1μg.

3. Incubation

   Transfected cells were incubated in CO₂ incubator for 72 hours.

4. Detection of β-gal expression
   1. Remove the medium from the well and wash the cells once with 100μl per well PBS.
   2. Fix the cells in the well with 50μl solution of 4% formaldehyde in PBS for 5min at room temperature.
   3. Wash each well twice with 100μl PBS.
   4. Add 50μl per well of substrate/stain solution and incubate the plate overnight at 37°C.
   5. Observe the cells under microscope and evaluate the proportion of blue (β-gal-positive) cells.