Cell Subculture Protocol

HEK293 are rounded cells that grow in suspension in cell culture (although, initially they were an adherent cell line). HEK cells should be grown in a complete SFMII growth medium supplemented with 4mM L-glutamine. Cells should be incubated at 37.0°C in 5% CO2 and expected to double approximately every 34 hours.

Subculture Protocol for HEK293:

Renew medium 2-3 times a week, and keep cell count between 1 x 10^5 and 3 x 10^5 cells/mL. Do no allow cell concentration to exceed 1 x 10 ^ 6 cells/mL

Cells can also be subcultured by centrifuging and resuspending in fresh medium:

  1. Transfer suspension to centrifuge tube
  2. Rap side of emptied flask to remove adherent cells
  3. Resuspend newly dislodged cells in 5 mL medium and aspirate with a small pipette to disperse clumps
  4. Pour resuspended cells into centrifuge tube with rest of suspension
  5. Centrifuge at 125xg for 5-7 minutes
  6. Remove supernatant and resuspend cells with fresh medium

To preserve:

Freeze cells in conditioned growth medium supplemented with 10% (v/v) DMSO. Store in the liquid nitrogen vapor phase or at -135 °C.


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HEK293 research articles:

  • Production of P-glycoprotein: HEK 293 cells were transfected to transiently express P-glycoprotein with a histidine tag to study the phenomenon of drug efflux. The P-glycoprotein was then purified with nickel-chelate chromatography and analyzed for drug binding and drug-stimulated ATPase activity. The results of this study suggested that drug-stimluated ATPase activity does not always match to the drug-resistant phenotype. [LINK]
  • Expression of μ-opioid receptor (μR): In this study, HEK 293 cells were used to express the μR molecule with an amino-terminal epitope tag. The tagged receptor was demonstrated to be comparable to the untagged receptor in binding of target ligands and inhibition of cyclic AMP accumulation. The tagged receptor was used to investigate the intracellular trafficking of the μR, and found that movement of the μR is agonist-dependent. [LINK]
  • Expression of G-protein coupled receptors (GPCRs): GPCRs are signaling proteins often produced in HEK 293 cells. In this study, microarrays were used to measure mRNA expression in a library of GPCR genes in several common GPCR-producing cell lines. The generation of this mRNA profile will aid future researchers in selection of the most appropriate cell line for their purposes, as well as elucidating interactions between GPCRs and related signaling molecules. [LINK]