Sep 06, 2022

Public workspaceCulture and transfection of HEK293T cells

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lynkzpvx9/v1
  • Hanqin Li1,
  • Dirk Hockemeyer1
  • 1University of California, Berkeley
Hanqin Li
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.eq2lynkzpvx9/v1
External link: https://doi.org/10.7554/eLife.79208
Protocol Citation: Hanqin Li, Dirk Hockemeyer 2022. Culture and transfection of HEK293T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynkzpvx9/v1
Manuscript citation:
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208

https://doi.org/10.7554/eLife.79208
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
We use this protocol and it's working
Created: July 23, 2022
Last Modified: December 13, 2024
Protocol Integer ID: 67395
Keywords: ASAPCRN, transfection, cell, lipofectamine
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This protocol describes a standard procedure culturing and transfecting HEK293T cells

Protocol overview:
A. Culturing HEK293T cells
B. Transfection of HEK293T cells with Lipofectamine 2000
Materials
ABC
Item Vendor Catalog #
DMEM, high glucose Thermo Fisher 11965118
DPBS w/o Calcium and magnesium Corning MT21031CV
Fetal Bovine Serum (FBS) Corning 35-011-CV
L-Glutamine Sigma G8540
MEM Non-Essential Amino Acids (100X) Thermo Fisher 11140050
0.25% Trypsin with EDTA Thermo Fisher 25200114
Opti-MEM Thermo Fisher 31985062
Sodium Pyruvate 100 mM Thermo Fisher 11360070
Lipofectamine 2000 Thermo Fisher 11668019
Troubleshooting
A. Culturing HEK293T cells
1m
HEK293T cells are cultured in HEK293T medium in 10 cm dishes.
HEK293T Medium
AB
DMEM, high glucose 385 ml
Fetal Bovine Serum (FBS) 50 ml
L-Glutamine (100X) 5 ml
MEM Non-Essential Amino Acids (100X) 5 ml
Sodium Pyruvate 100 mM5 ml
Final volume: 500 mL
Passage cells when the culture reaches 80% confluency.
Remove medium
Wash once with 5 ml DPBS
Add 1 ml 0.25% Trypsin with EDTA, tilt and shake the dish so that the Trypsin covers the entire dish.
Incubate at Temperature37 °C for Duration00:01:00

1m
Add 5 ml fresh HEK293T medium to inactivate Trypsin
Pipet 10 times to dissociate the cells and mix
Transfer 1 ml of cell suspension to a new dish pre-added with 9 ml HEK293T medium. Shake to mix well.This is a 1:6 splitting.
HEK293T cells usually needs to be passaged every 2 days.
B. Transfection of HEK293T cells with lipofectamine 2000
45m
One day before transfection, dissociate HEK293T cells with Trypsin as described above
Seed 250,000 cells/1 well of 12-well plate
Change to fresh medium 1 h before transfection
Label two micro centrifuge tubes as I and II
In micro centrifuge tube I, add 125 µl Opti-MEM and 6 µl Lipofectamine 2000. Mix by gently pipetting 3 times. Incubate at TemperatureRoom temperature for Duration00:05:00 .

5m
While waiting, in micro centrifuge tube II, add 125 µl Opti-MEM and 250 ng total plasmid. Mix by pipetting.
Mix tube I and II by gently pipetting 3 times. Incubate at TemperatureRoom temperature for Duration00:20:00 .

20m
Mix one time by gently pipetting. Transfer all transfection reagents into one well of a 12-well plate, drop-wise.
After adding the reagent to all wells, shake the plate to mix.
Culture in Temperature37 °C incubator DurationOvernight .

20m
Change to fresh medium and culture for another 2 days. Collect samples or passage once if longer culturing is needed.