X-Gal staining of HeLa (Cervical Adenocarcinoma) cells 24 hours after transfection in 96 well plates

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All Complexes were formed in OptiMEM®.


HeLa 10,000/well

Reagent / DNA Ratio
5μl Complex per well
Complex Incubation 10 min

Lipofectamine® LTX

Reagent / DNA Ratio
20μl Complex per well
Complex Incubation 30 min

Lipofectamine® 2000

Reagent / DNA Ratio
50μl Complex per well
Complex Incubation 20 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 1×103 and 3×103 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 24 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.