X-Gal staining of the Mouse Embryonic Stem Cells After Transfection by FuGENE®HD, FuGENE®6 and Competitor Lipofectamine® 2000. Embryonic stem cells were grown on STO-mytomycin C treated fibroblasts (STO-1) in T25 flasks

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FuGENE®HD

 


Reagent / DNA Ratio
15μl/5μg
265μl Complex per flask
Complex Incubation 15 min

Lipofectamine® 2000


Reagent / DNA Ratio
20μl/8μg
1000μl Complex per flask
Complex Incubation 20 min

FuGENE®6

 


Reagent / DNA Ratio
15μl/5μg
500μl Complex per flask
Complex Incubation 15 min
Transfection with competitor reagent was performed according to manufacturer’s instructions

Protocol for Mouse Embryonic Stem Cell

  1. Cell plating

    ES cells (from Celprogen, Inc) were seeded the day before transfection with the density 10,000 cells per well in 100 μl complete growth medium Mouse Embryonic Stem Cell Expansion (Celprogen, Inc).

  2. Complex preparation

    Tissue culture 48 flat-bottom well plates were used for complex preparation:

    1. Prepare 0.02μg/μl pCMVβ DNA solution in OptiMEM®.
    2. Add 30μl of reagent to 500 μl of OptiMEM® /DNA solution.
    3. Mix carefully by pipetting (10-15 times).
    4. Incubate 15 min at room temperature for complex formation.
    5. Add 265μl complex per T25 flask to the cells, and mix thoroughly.
  3. Incubation

    Place the cells into CO2 incubator for 24 hours.

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