HEK293A GFP-LC3

Cell Line Description

A stable cell line expressing EGFP-tagged LC3 (rat) in background of HEK293 cell line. This reporter cell line can be used to monitor induction of autophagy after amino acid starvation or rapamycin treatment as well as autophagosome formation. Also, this cell line provides a useful tool for the study of autophagy using GFP-LC3 as a standard assay read out by both immunofluorescence and biochemical methods.

To generate the cell line EGFP-LC3 (rat) plasmid was transfected into HEK293A cells. The cells were selected with 800ug/ml Geneticin. Single cell clones were selected and grown up. Several clones were tested and clone 2GL9 was taken as the best based on a number of parameters (including GFP-LC3 expression levels and responsiveness to amino acid deprivation).

Description of the genetic modification to generate this cell line: HEK293 cells transfected with a GFP-LC3 (green fluorescent protein-microtubule-associated protein 1 light chain 3) plasmid. cDNA encoding rat LC3 was obtained by RT–PCR from rat brain total cDNA and subcloned into the EcoRI site of the eukaryotic expression vector pCI-neo (Promega, Madison, WI). It was then excised and the LC3 cDNA inserted into the BglII and EcoRI sites of pEGFP-C1, a GFP fusion protein expression vector (Clontech Laboratories), Kabeya Y, et.al. (2000).

Culture Medium

Dulbecco′s Modified Eagle′s Medium, high glucose (DMEM) + 2mM Glutamine + 10% Fetal Bovine Serum (FBS).

Geneticin at (400ug/ml) can be added for maintenance but this is not normally necessary as the GFP-LC3 expression level is maintained over at least 20 passages.

Subculture Routine

Trypsinise cultures at 80% confluence with 0.05% Trypsin/EDTA. Seed flasks at 2 x 10⁴ cells/ cm². Cultures must be incubated in a humidified 10% CO₂/90% air incubator at 37^oC.

Other Notes

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Cultures from PHE Culture Collections and supplied by Sigma are for research purposes only. Enquiries regarding the commercial use of a cell line are referred to the depositor of the cell line. Some cell lines have additional special release conditions such as the requirement for a material transfer agreement to be completed by the potential recipient prior to the supply of the cell line. Please view the Terms & Conditions of Supply for more information.

Disclaimer

This cell line has special release conditions: Commercial organisations are required to complete the ′Cell Line Release Authorisation for Research Use in Commercial Organisations′ release conditions form.