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X-Gal Staining of Caki-1 (Human kidney clear cell carcinoma) cells 48 hours after transfection by FuGENE®HD and Reagents from Competitors in 96 well plates, 10,000cells/well.

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Transfection is done in 100% Fetal Bovine Serum Transfection is done in growth medium, McCoy’s 5A medium+10% Fetal Bovine Serum

Reagent / DNA Ratio
0.3μl/0.1μg
5μl Complex per well
Complex Incubation 10 min		 Complexes are formed in OptiMEM®

FuGENE® HD

 
Reagent / DNA Ratio
0.3μl/0.1μg
5μl Complex per well
Complex Incubation 10 min

Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min		 Complexes are formed in OptiMEM®

Lipofectamine® 2000

 
Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min

Reagent / DNA Ratio
0.3μl/0.1μg
20μl Complex per well
Complex Incubation 30 min		 Complexes are formed in OptiMEM®

Lipofectamine® LTX

 
Reagent / DNA Ratio
0.3μl/0.1μg
20μl Complex per well
Complex Incubation 30 min

Reagent / DNA Ratio
0.5μl/0.1μg
20μl Complex per well
Complex Incubation 20 min		 Complexes are formed in 0.15M NaCl

JET.PEI™

 
Reagent / DNA Ratio
0.5μl/0.1μg
20μl Complex per well
Complex Incubation 20 min
Transfection with competitor reagent was performed according to manufacturers’ instructions

Protocol for Transfection of Caki-1, (ATCC®: HTB-46™ Human Kidney Carcinoma) Cells by FuGENE HD in 96 well plates.

  1. Cell plating

    Caki-1 cells were seeded from 90-100% confluent culture the day before transfection with the density 10,000 cells/well in 100 μl complete growth medium (McCoy’s 5A Medium + 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.

  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 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 reagent/DNA may vary in the range 0.25μl/0.1μg to 0.4μl/0.1μg.

  3. Incubation

    Transfected cells were incubated in CO2 incubator for 48 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.