Procedure for colony assay on coated coverslips:

1

• Procedure for colony assay on coated coverslips
• Plated 2000 EpH4 cells on top of coverslips in
  6-well dishes (normal growth medium).
• After 3 days in culture, rinsed coverslips w/ PBS
• Fixed in 2 paraformaldehyde in PBS for 20 min
• Stopped with PBS/Glycine 1hr.
• Blocked in IF wash 1hr
• Incubated face-down on top of 50uL of primary
  antibody solution (in IF wash) on Parafilm O/N _at_
  4C in humidified chamber.
• Rat anti-cytokeratin 8 (DSHB TROMA-I, 14
  supernatant)
• Rabbit anti-cytokeratin 14 (Covance PRB155P,
  11000)
• Washed 3x in IF wash for 20min each.
• Incubated face-down on top of 50uL of secondary
  antibody solution on Parafilm 20min _at_ RT
• Goat anti-rat FITC (Jackson, 1200)
• Goat anti-rabbit TR (Jackson, 1200)
• Washed 2x in IF wash for 10min each
• Incubated w/ DAPI (5ug/mL in IF wash) 5 min
• Washed O/N _at_ 4 C in IF wash.
• Mounted on slides w/ Vectamount, sealed w/ nail
  polish.

2

• 22mm No.1 coverslips were coated as follows
• Poly-D-Lysine
• Piled 400uL working dilution (20ug/mL in PBS) on
  top of coverslip.
• Incubated 2hrs _at_ RT in laminar hood.
• Removed excess and washed 5x with H2O for 5 min
  each.
• Laminin (purified mouse Laminin1)
• First coated with PDL as outlined above
  (increases protein binding)
• Placed 100uL working dilution (20ug/mL in PBS) on
  Parafilm.
• Incubated coverslip face-down on top of Laminin
  in humidified chamber (wet filter paper under
  parafilm, sealed cover) _at_ 4C overnight.
• Remove excess, rinse once w/ PBS before plating
  cells on top.
• Fibronectin
• First coated w/ PDL.
• Placed 100uL working dilution (1ug/mL in PBS
  (Ca2/Mg2-free)) on parafilm.
• Incubated face-down on top of fibronectin _at_ 4C
  O/N in humidified chamber
• Removed excess, rinsed once w/PBS.

3
Solutions

• 10x PBS/Glycine
• 38g NaCl
• 9.38g Na2HPO4
• 2.07g NaH2PO4
• 37.5g Glycine
• To 500mL in dH2O, sterile filtered, pH 7.4
• 10x IF Wash
• 38g NaCl
• 9.38g Na2HPO4
• 2.07g NaH2PO4
• 2.50g NaN3
• 5.00g BSA
• 10.00mL Triton-X 100
• 2.50mL Tween-20
• To 500mL in dH2O, sterile filtered, pH 7.4

4
Phenotypes of colonies forming from single cells
on top of different substrates.
Glass n 18 PDL n 18 Laminin n
22 Fibronectin n 23
5
Phenotypes of colonies forming from single cells
on top of different substrates.
Glass n 18 PDL n 18 Laminin n
22 Fibronectin n 23
6
Examples of clustered and scattered colonies.
7
Examples of colonies arising from single cells
Fixed and stained against K8 (FITC) and K14 (TR),
nuclei (DAPI)
Colonies at Day 2 before fixing
K8 colony
1 hour after plating
Mixed colony
8
Example of colonies arising from multiple cells
9
Phenotypes of colonies forming from single or
multiple cells on top of coverslips.
Single cells n 35 2-3 cells n 18