Jun 23, 2016
Version 2
Protocol for STO Cell Transfection by FuGENE HD V.2
- Misha Gurevich1,
- V. Katerov1
- 1Fugent LLC
Protocol Citation: Misha Gurevich, V. Katerov 2016. Protocol for STO Cell Transfection by FuGENE HD. protocols.io https://dx.doi.org/10.17504/protocols.io.e8sbhwe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 23, 2016
Last Modified: November 09, 2017
Protocol Integer ID: 3058
Abstract
Protocol for Transfection Mouse Embryonic Stem Cells.
Cell plating
Cell plating
STO cells were seeded the day before transfection with the density 15,000 cells per well in 100 μl complete growth medium DMEM+10% Fetal Bovine Serum.
Complex preparation (per 20 wells)
Complex preparation (per 20 wells)
Prepare 0.02μg/μl pCMVβ plasmid DNA solution in OptiMEM®.
Note
Tissue culture 96-round bottom well plates were used for complex preparation.
Add 6μl of reagent to 100 μl of OptiMEM® /DNA solution.
Mix carefully by pipetting (10-15 times).
Incubate 5 min at room temperature.
00:05:00
Add 5μl complex per well to the cells, and mix thoroughly.
Note
Optimal ratio reagent/DNA may vary in range 0.2μl/0.1μg to 0.35μl/0.1μg.
Incubation
Incubation
Place the cells into CO2 incubator for 26-28 hours.
26:00:00
Detection of β-gal expression
Detection of β-gal expression
Remove the medium from the well and wash the cells once with 100μl per well PBS.
Fix the cells in the well with 50μl solution of 4% formaldehyde in PBS for 5min at room temperature.
00:05:00
Wash each well with 100μl PBS. (1/2)
Wash each well with 100μl PBS. (2/2)
Add 50μl per well of substrate/stain solution and incubate the plate overnight at 37°C.
16:00:00
Observe the cells under microscope and evaluate the proportion of blue (β-gal-positive) cells.