Plate Format:	96 well
Plate Coating:	Poly-D-Lysine
Comments:	Delicate cell line, prone to loss by washing.

HEK 293

Transgene:	LC3-GFP
Origin:	Tooze lab
Cell Medium:	E4 + 10% FCS
Cell Seeding Density Per Well:	4000

Protocol:	Mix siRNA in 10uL HBSS to 37.5nM final concentration. Mix 0.4uL Lipofectamine2000 in 10uL OPTIMEM. Mix together on plate and leave for 20 minutes. Add cells in 80uL E4 medium with 10%FBS, pen/strep and supplemented with L-glutamine. Change into fresh media for 16-24 hours.
Delivery Reagent:	Lipofectamine 2000
Delivery Reagent Cat No:	11668-019
Delivery Reagent Supplier:	Invitrogen
Delivery Reagent Batch No:	Lot394876
Delivery Reagent Concentration:	0.4ul/well
Silencing Reagent Concentration:	37.5nM
Silencing Reagent Supplier:	Dharmacon
Silencing Reagent Origin:	HTS lab

• Induction with EBSS and leupeptin
  Treatment:Wash plates twice to remove medium. Starve cells in amino acid-free medium(EBSS) supplemented with leupeptin (0.25mg.mL). 2 hours.
  Compounds:Leupeptin, Earls Buffered Salt Solution (EBSS)
  Compound Concentration:0.25mg/ml
  Treatment Time:2 hours
  

Description:Fixed in 4% formaldehyde for 30mins
Fixation Compounds:Formaldehyde
Compound Concentration:4%
Treatment Time:30mins

Nuclear Staining:DAPI
Nuclear Staining Concentration:1ug/ml
Nuclear Staining Incubation Time:1 hour

• Cellomics ArrayScan Ch1
  Localisation:nucleus
  Stain:DAPI
  
• Cellomics ArrayScan Ch2
  Localisation:whole cell
  Stain: LC3-GFP