Oct 17, 2025
Tissue culture protocol for HEK293 cells
- Susanne Herbst1,2,
- Patrick Lewis1,2
- 1RVC;
- 2The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Protocol Citation: Susanne Herbst, Patrick Lewis 2025. Tissue culture protocol for HEK293 cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zy7dgqe/v1
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: October 17, 2025
Last Modified: October 17, 2025
Protocol Integer ID: 230072
Keywords: ASAPCRN, Tissue culture, hek293 cells lewis lab tissue culture protocol, hek293 cell, tissue culture protocol
Funders Acknowledgements:
ASAP
Abstract
Lewis lab tissue culture protocol for HEK293 cells.
Materials
- DMEM (31966047, Gibco)
- heat-inactivated FCS
- Trypsin-EDTA (25300054, Gibco)
- PBS
- DMSO
Troubleshooting
Subculturing
Remove the medium and add 5 ml of PBS to 75 cm^2 flask.
Remove PBS and add 3 ml of Trypsin-EDTA (25300054, Gibco).
Place flask back in incubator for 2 min.
Check if cells have detached, then add 7 ml of complete medium.
Aliquot cell suspension into new flask at a ratio of 1:3 to 1:5.
Incubate the culture at 37°C, 5% CO2 in a suitable incubator.
Subculture every 2 to 3 days.
Thawing
Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol.
Centrifuge the cell suspension at approximately 300 x g for 5 minutes. Discard the supernatant and resuspend the cell pellet in an appropriate amount of fresh growth medium and transfer the cells to an appropriate size vessel.
Incubate the culture at 37°C, 5% CO2 in a suitable incubator.
Freezing
Freeze cell suspension in complete growth medium supplemented with 10 % (v/v) DMSO at 1 to 5 x 10^6 cells/ml.