3. Anti-Snail1 (Cell Signaling Technology 3879S).
4. Anti-Fibronectin (Agilent DAKO A0245).
5. Anti-Vimentin (Agilent DAKO M0725).
6. Set of anti-Rabbit and anti-Mouse secondary antibodies coupled with fluorophores.
7. 32% paraformaldehyde aqueous solution (Electron Microscopy
Sciences).
8. PBS solution (Gibco or any equivalent reference from other
suppliers).
9. Triton X-100 aqueous solution (Merck or any equivalent reference from other suppliers).
10. Foetal Bovine Serum (FBS).
11. Bovine Serum Albumin (BSA).
12. Diamidino-2-phenylindole (DAPI) solution (1 mg/mL)
(ThermoFisher) or any equivalent reference from other
suppliers.
3 Methods
This protocol consists of the repetition of 4 cycles of EMT/MET
(Fig. 1a). At each half cycle, we characterize the cells for epithelial
and mesenchymal properties (Fig. 1b).
3.1 Induction of the
First EMT
1. For immunofluorescence labelling, place coverslips in 60 mm
culture dishes and coat with a 0.1% gelatine/PBS solution. See
Note 4 to manage the number of dishes to prepare.
2. Seed MCF10A cells at a density of 400,000 cells per 60 mm
culture dish (see Notes 4 and 5) in control medium and keep in
a 5% CO 2 atmosphere at 37
C.
3. 12 hs after (day 0), replace the control medium by the
EMT-IM.
4. After 48 h, replace EMT-IM by the control medium and keep
cells in culture for an additional 48 h.
3.2 Induction of the
First MET
1. At day 4 (96 h after the beginning of the EMT induction),
photograph cells are using a phase-contrast inverted
microscope.
2. Recover coverslips and proceed for immunofluorescence
labelling.
3. Dissociate the other cells using Accutase, and seed 400,000
cells per 60 mm culture dish in control medium (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).
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
345
4. Anti-Fibronectin (Agilent DAKO A0245).
5. Anti-Vimentin (Agilent DAKO M0725).
6. Set of anti-Rabbit and anti-Mouse secondary antibodies coupled with fluorophores.
7. 32% paraformaldehyde aqueous solution (Electron Microscopy
Sciences).
8. PBS solution (Gibco or any equivalent reference from other
suppliers).
9. Triton X-100 aqueous solution (Merck or any equivalent reference from other suppliers).
10. Foetal Bovine Serum (FBS).
11. Bovine Serum Albumin (BSA).
12. Diamidino-2-phenylindole (DAPI) solution (1 mg/mL)
(ThermoFisher) or any equivalent reference from other
suppliers.
3 Methods
This protocol consists of the repetition of 4 cycles of EMT/MET
(Fig. 1a). At each half cycle, we characterize the cells for epithelial
and mesenchymal properties (Fig. 1b).
3.1 Induction of the
First EMT
1. For immunofluorescence labelling, place coverslips in 60 mm
culture dishes and coat with a 0.1% gelatine/PBS solution. See
Note 4 to manage the number of dishes to prepare.
2. Seed MCF10A cells at a density of 400,000 cells per 60 mm
culture dish (see Notes 4 and 5) in control medium and keep in
a 5% CO 2 atmosphere at 37
C.
3. 12 hs after (day 0), replace the control medium by the
EMT-IM.
4. After 48 h, replace EMT-IM by the control medium and keep
cells in culture for an additional 48 h.
3.2 Induction of the
First MET
1. At day 4 (96 h after the beginning of the EMT induction),
photograph cells are using a phase-contrast inverted
microscope.
2. Recover coverslips and proceed for immunofluorescence
labelling.
3. Dissociate the other cells using Accutase, and seed 400,000
cells per 60 mm culture dish in control medium (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).
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
345