Cell culture

Elias Adriaenssens

Published: 2023-09-23 DOI: 10.17504/protocols.io.n2bvj3y5blk5/v1

Abstract

This protocol describes routine cell culture maintenance.

Steps

Cell culture

1.

Grow HeLa and HEK293T cells in Dulbecco Modified Eagle Medium (DMEM, Thermo

Fisher) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Thermo Fisher),25millimolar (mM) HEPES (Thermo Fisher), 1% (v/v) non-essenMal amino acids (NEAA, Thermo Fisher), and 1% (v/v) Penicillin-Streptomycin (Thermo Fisher).

2.

Culture HAP1 cells in Iscove’s Modified Dulbecco’s Medium (IMDM, Thermo Fisher), supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Thermo Fisher) and 1% (v/v) Penicillin-Streptomycin (Thermo Fisher).

3.

Regularly test all cell lines for mycoplasma contaminations.

4.

Split the cells by trypsinization once confluent.

5.

To this end, remove medium with Pasteur pipette.

6.

Wash cells with 1x PBS and then add trypsin for 0h 5m 0s at 37°C.

7.

Afterwards, resuspend cells in medium and retain 10% for further culturing and expansion.

8.

Discard the rest or collect for seeding for experiments.

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