6. Wash with 200 μL staining media. Repeat for a total of three
times.
7. Resuspend cells in 100 μL cold-staining buffer, add 1 μL
(1:100) Brilliant Violet 421 secondary antibody and incubate
on ice for 15 min in the dark. Add secondary antibody to
unstained control.
8. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant. Wash with 200 μL staining media. Repeat for a
total of three times.
9. Fix cells by adding 200 μL intracellular fixation buffer (dilute
1-part fixation/permeabilization concentrate with 3 parts fixation/permeabilization diluent) and incubate 30 min at room
temperature. Cover plate with aluminum foil or store in
drawer.
10. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant.
11. Add 100 μL 1Â Permeabilization Buffer (10Â permeabilization buffer diluted tenfold in distilled water).
12. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant.
13. Resuspend in 100 μL 1Â permeabilization buffer with 1:250
rat anti-ECAD primary antibody and incubate on ice for
15 min.
14. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant. Wash three times with 1Â permeabilization
buffer.
15. Resuspend cells in 100 μL 1Â permeabilization buffer, add
1 μL (1:100) APC anti-rat secondary antibody, and incubate
on ice for 15 min in the dark. Add secondary antibody to
unstained control. Single color controls should be run as well
(see Note 13).
16. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant. Wash three times with staining buffer.
17. Resuspend cells with 100 μL staining buffer and analyze by
flow cytometry. Tumor cells that are in a partial EMT will be
Brilliant Violet E-cadherin negative for surface expression but
APC E-cadherin positive for intracellular expression. Strategy
for detecting intracellular proteins are shown in Fig. 2d of
Aiello et al. [6].
324
Robert J. Norgard and Ben Z. Stanger
times.
7. Resuspend cells in 100 μL cold-staining buffer, add 1 μL
(1:100) Brilliant Violet 421 secondary antibody and incubate
on ice for 15 min in the dark. Add secondary antibody to
unstained control.
8. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant. Wash with 200 μL staining media. Repeat for a
total of three times.
9. Fix cells by adding 200 μL intracellular fixation buffer (dilute
1-part fixation/permeabilization concentrate with 3 parts fixation/permeabilization diluent) and incubate 30 min at room
temperature. Cover plate with aluminum foil or store in
drawer.
10. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant.
11. Add 100 μL 1Â Permeabilization Buffer (10Â permeabilization buffer diluted tenfold in distilled water).
12. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant.
13. Resuspend in 100 μL 1Â permeabilization buffer with 1:250
rat anti-ECAD primary antibody and incubate on ice for
15 min.
14. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant. Wash three times with 1Â permeabilization
buffer.
15. Resuspend cells in 100 μL 1Â permeabilization buffer, add
1 μL (1:100) APC anti-rat secondary antibody, and incubate
on ice for 15 min in the dark. Add secondary antibody to
unstained control. Single color controls should be run as well
(see Note 13).
16. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant. Wash three times with staining buffer.
17. Resuspend cells with 100 μL staining buffer and analyze by
flow cytometry. Tumor cells that are in a partial EMT will be
Brilliant Violet E-cadherin negative for surface expression but
APC E-cadherin positive for intracellular expression. Strategy
for detecting intracellular proteins are shown in Fig. 2d of
Aiello et al. [6].
324
Robert J. Norgard and Ben Z. Stanger