X-Gal staining of Human Skeletal Muscle Myoblast cells 48 hours after transfection by FuGENE®HD and Reagents from Competitor in 96 well plates. 3,000 cells/well.

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All complexes are formed in OptiMEM®.

FuGENE®HD

 


Reagent / DNA Ratio
0.225μl/0.075μg
3.75μl Complex per well
Complex Incubation 10 min

Lipofectamine® 2000

 


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

Lipofectamine® LTX

 


Reagent / DNA Ratio
0.25μl/0.1μg
20μl Complex per well
Complex Incubation 30 min
Transfection with competitor reagents was performed according to manufacturer’s instructions

Protocol for Human Skeletal Muscle Myoblasts (HSMM) Transfection by FuGENE HD

  1. Cell plating

    HSMM (Cambrex Bio Science Walkersville, Inc.) cells were seeded the day before transfection with the density 3,000 cells per well in 100 μl SkBM-2 basal growth medium supplemented with SkGM-2 BulletKit™ (Cambrex Bio Science Walkersville, Inc.).

  2. Complex preparation (per 20 well)

    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 (10 times).
    4. Incubate complex for 10 min. at room temperature.
    5. Add 3.75 μl of complex per well to the cells, and mix thoroughly.
  3. Incubation

    Place the cells into 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.