| 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 |
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:
- Channel 1: DAPI
- Channel 2: LC3-GFP
