Plate Format:

384 well

RPE1

Origin:	Cell Services
Cell Medium:	DMEM + 10% heat inactivated FCS +1% P/S + 1% Non-Essential Amino Acids
Cell Seeding Density Per Well:	900

Protocol:	Mix siRNA in 2.5uL HBSS to 375nM concentration. Mix 0.1uL Lullaby in 2.5uL OPTIMEM. Mix together on plate and leave for 20 minutes. Add cells in 45ul/well. Culture the cells for further 72hours. 54h after transfection cells are treated with ICRF193 and nocodazole for 17h.
Delivery Reagent:	Lullaby
Delivery Reagent Supplier:	OZ biosciences
Delivery Reagent Concentration:	0.1µl/well
Silencing Reagent Concentration:	18.75nM
Silencing Reagent Supplier:	Dharmacon
Silencing Reagent Origin:	HTS Lab

• Assessment of the G2 catenation checkpoint
  Treatment:add 50µl fresh media plus drugs
  Compounds:ICRF193/nocodazole
  Compound Concentration:3µM ICRF193, 1µM Nocodazole
  Treatment Time:16-18h
  
• Rehydration
  Treatment:Aspirate fixative and add PBS, leave for 1h
  

Description:fixed in solvent overnight
Fixation Compounds:Ethanol/Acetic acid
Compound Concentration:95/5 Ethanol 96%/Glacial acetic acid
Treatment Time:long-term at -20C

Description:Staining of mitotic cells
Blocking:2.5% BSA/PBS
Permeabilisation:none

• Primary Antibody - MPM2-Cy5
  Supplier:Millipore
  Cat No:16-220
  Batch No:DAM1768668
  Dilution:1:2000
  Incubation Time:2h
  
• Secondary Antibody - Anti-Mouse PacificGreen
  Supplier:Invitrogen
  Dilution:1:1000
  Incubation Time:2h
  

Nuclear Staining:DAPI
Nuclear Staining Concentration:1ug/ml
Nuclear Staining Incubation Time:2 hour

• Cellomics ArrayScan Ch1
  Localisation:Nucleus
  Stain:DAPI
  
• Cellomics ArrayScan Ch2
  Localisation:Cytosol
  Stain:MPM2-Cy5
  
• TTP Acumen Explorer Ch1
  Localisation:Nucleus
  Stain:DAPI
  
• TTP Acumen Explorer Ch2
  Localisation:Nucleus
  Stain:MPM2-Pacific Green