4. After 48 h, change control medium and maintain cells in culture for an additional 48 h.
5. At day 8, (96 h after the beginning of MET induction), photograph cells using a phase-contrast inverted microscope.
Recover coverslips and proceed for immunofluorescence
labelling.
6. Dissociate the other cells using Accutase and seed 400,000 cells
per 60 mm culture dish in EMT-IM (see Notes 4 and 5). Keep
the remaining cells for gene and protein expression studies,
flow cytometry analysis or any other downstream application
(see Note 4).
3.3 Repetitions of
EMT/MET Cycles
Repeat steps of Subheadings 3.1 and 3.2 to reach the desired
number of EMT/MET cycles (see Note 6).
3.4 Quality Control of
EMT/MET Cycle Using
Phase-Contrast and
Fluorescent
Microscopy
1. At the end of each EMT or MET phases, remove the cover slips
and fix cells using a solution of 1% paraformaldehyde/PBS for
10 min at room temperature.
2. Wash cells twice with ice-cold PBS and permeabilize for 20 min
at 4
C using a 0.1% Triton X-100 PBS solution.
3. Block nonspecific antigen binding sites with a 0.1% Triton
X-100, 1% FBS PBS blocking solution for 1 h at room
temperature.
4. Incubate cells with anti-β-Catenin (using a 1/200 dilution in
blocking solution), anti-Fibronectin (using a 1/500 dilution in
blocking solution), anti-Snail1 (using a 1/400 dilution in
blocking solution) and anti-Vimentin (using a 1/500 dilution
in blocking solution) antibodies overnight at 4
C (see Note 7).
5. Wash twice with PBS and incubate cells at room temperature
with secondary antibodies for 60 min (using a 1/1000 dilution
in blocking solution) and stain the nuclei with DAPI (1 μg/mL
in PBS) for 10 min then wash with PBS twice.
6. Fix the stained cover slips onto glass slides using Dako fluorescence mounting medium and store away from light at 4
C.
7. Image cells using an epifluorescence microscope equipped with
phase contrast (Fig. 2).
ä
Fig. 2 (continued) Immunofluorescence labelling for prototypic epithelial and mesenchymal markers confirms
the transitions between both states. Epithelial cells are characterized by a clear localization of β-CATENIN at
adherent junction, low levels of FIBRONECTIN and VIMENTIN and the absence of SNAIL1. Mesenchymal cells
are characterized by a cytoplasmic re-localization of β-CATENIN, a strong expression of FIBRONECTIN with an
extra-cellular deposition of FIBRONECTIN fibres, a stronger expression of VIMENTIN and nuclear detection of
SNAIL1 in some cells
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
347
5. At day 8, (96 h after the beginning of MET induction), photograph cells using a phase-contrast inverted microscope.
Recover coverslips and proceed for immunofluorescence
labelling.
6. Dissociate the other cells using Accutase and seed 400,000 cells
per 60 mm culture dish in EMT-IM (see Notes 4 and 5). Keep
the remaining cells for gene and protein expression studies,
flow cytometry analysis or any other downstream application
(see Note 4).
3.3 Repetitions of
EMT/MET Cycles
Repeat steps of Subheadings 3.1 and 3.2 to reach the desired
number of EMT/MET cycles (see Note 6).
3.4 Quality Control of
EMT/MET Cycle Using
Phase-Contrast and
Fluorescent
Microscopy
1. At the end of each EMT or MET phases, remove the cover slips
and fix cells using a solution of 1% paraformaldehyde/PBS for
10 min at room temperature.
2. Wash cells twice with ice-cold PBS and permeabilize for 20 min
at 4
C using a 0.1% Triton X-100 PBS solution.
3. Block nonspecific antigen binding sites with a 0.1% Triton
X-100, 1% FBS PBS blocking solution for 1 h at room
temperature.
4. Incubate cells with anti-β-Catenin (using a 1/200 dilution in
blocking solution), anti-Fibronectin (using a 1/500 dilution in
blocking solution), anti-Snail1 (using a 1/400 dilution in
blocking solution) and anti-Vimentin (using a 1/500 dilution
in blocking solution) antibodies overnight at 4
C (see Note 7).
5. Wash twice with PBS and incubate cells at room temperature
with secondary antibodies for 60 min (using a 1/1000 dilution
in blocking solution) and stain the nuclei with DAPI (1 μg/mL
in PBS) for 10 min then wash with PBS twice.
6. Fix the stained cover slips onto glass slides using Dako fluorescence mounting medium and store away from light at 4
C.
7. Image cells using an epifluorescence microscope equipped with
phase contrast (Fig. 2).
ä
Fig. 2 (continued) Immunofluorescence labelling for prototypic epithelial and mesenchymal markers confirms
the transitions between both states. Epithelial cells are characterized by a clear localization of β-CATENIN at
adherent junction, low levels of FIBRONECTIN and VIMENTIN and the absence of SNAIL1. Mesenchymal cells
are characterized by a cytoplasmic re-localization of β-CATENIN, a strong expression of FIBRONECTIN with an
extra-cellular deposition of FIBRONECTIN fibres, a stronger expression of VIMENTIN and nuclear detection of
SNAIL1 in some cells
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
347