Day 1: Transfection of HT1080 cells stably expressing AP-HB-EGF

HT1080-AP-HB-EGF cells were reverse tranfected with siRNA using INTERFERin transfection reagent in 96-well plates.

Day 3: TPA induction / media change and cell fixation

Plates were washed twice with PBS and treated with 400nM TPA or Serum Free Media (control) for 30mins at 37C.

Media was then removed and cells were fixed in 4% formaldehyde. 

Weeks 2 - 6: Immunofluorescent staining of cell surface expression of AP-HBEGF

Plates were stained with:

• DAPI
• Anti-alkaline phosphatase monoclonal antibody (clone 8B6 from Sigma)

Readout

Acumen Explorer eX3 laser scanning cytometer was used to determine cell number and cell surface expression of AP-HBEGF.