Screen Status: Primary
Screen Overview: Genome screen of HEK293 cells expressing the GFP-LC3 marker for autophagy. Cells are cultured in amino acid deprived medium to induce starvation mediated autophagy. Results are gained by counting spots which represent autophagosomes.
Screener: Nicole McKnight (Tooze Lab).
Extension:3145, Rm 612
Library: Full Genome 96
Library Description: 270 plates consisting of Kinome, GPCR, druggable and genome libraries
Library Supplier: Dharmacon
Library Format: 267 plate(s) in 96 well format
Replicate Info: 3 replicates
Publications: Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC.. McKnight et al., 2012
Keywords: Amino acid deprivation, Autophagosome, Autophagy
Cell Line: HEK 293

Screen Method

Day 1: Transfection of Hek293 GFP LC3 cells

Hek293 GFP LC3 cells were reverse transfected with siRNA using Lipofectamine 2000 transfection reagent in 96 well plate format.

 

Day 3: Media change

Culture media was removed and fresh full media was added to alllow the cells recover from the transfection stress. Plates then incubate overnight at 37°C.

 

Day 4: EBSS induction and cell fixation

Plates were washed with PBS and then incubated EBSS+Leupeptin for 2 hours at 37C.

Cells were then fixed with 4% formaldehyde.

Day 5: Cell staining

Plates were stained with DAPI.

Images were collected on ArrayScan microscope: