Assay Overview

Plate Format: 384 well

Assay Cell Culture

HaCaT

Origin: Hill Lab
Cell Medium: DMEM+10%FCS+1%PenStrep
Cell Seeding Density Per Well: 2000

Delivery Protocol - Reverse/Wet transfection

Protocol: Mix siRNA in 2.5uL HBSS to 375nM concentration. Mix 0.1uL Interferin in 2.5uL OPTIMEM. Mix together on plate and leave for 20 minutes. Add cells in 20ul/well. Top-up 25ul fresh media per well after 24hour. Culture the cells for further 48hours
Delivery Reagent: Interferin
Delivery Reagent Cat No: 409-10
Delivery Reagent Supplier: Polyplus(via SourceBioscience)
Delivery Reagent Batch No: 051NF0204G6
Delivery Reagent Concentration: 0.1ul/well
Silencing Reagent Concentration: 37.5nM (b.f. 24hour)
Silencing Reagent Supplier: Dharmacon
Silencing Reagent Origin: HTS Lab

Assay Treatments

Cell Fixation

Fixed in 4% formaldehyde for 30mins
Formaldehyde
4%
15min-30min

Assay Staining

Smad2/3 staining
3%BSA/10%FCS/0.3%TritonX100/PBS
1hour
DAPI
1ug/ml
1 hour

Assay Readouts