12. Centrifuge cells at 4
C at 300 RCF for 5 min and discard
supernatant. Wash pellet with 500 μL staining media. Repeat
for a total of three times.
13. Resuspend cells in 500 μL ice cold-staining buffer, add 5 μL
(1:100) APC donkey anti-rat secondary antibody and incubate
on ice for 15 min in the dark. Add 180 μL ice cold-staining
buffer to the 20 μL unstained negative control sample, and add
2 μL secondary antibody (1:100).
14. Centrifuge cells at 4
C at 300 RCF for 5 min and discard
supernatant. Wash with 500 μL staining media. Repeat for a
total of three times including negative control.
15. Resuspend cells in 1.5 mL sorting/staining buffer with 1.5 μL
Dapi (1:1000). Use an 18-gauge needle in a 3 mL syringe to
aspirate the cell solution. Remove the needle and then eject the
solution into a polystyrene round-bottom tubes with 35 μm
cell strainer cap. Depending on the type of FACS machine
being used, polypropylene round-bottom tubes may need to
be used. The filter cap can be switched onto these tubes or the
cells can be transferred into this type of tube. Keep the cells on
ice and dark until use.
16. Sort the cells in a FACs machine.
17. When processing using FACS, viable cells are impermeable to
DAPI, so DAPI positive cells are no longer viable and excluded
from the sort. It is important to use staining buffer without
phenol red, as this may interfere with fluorescent signal. Epithelial cells with be positive for YFP and E-cadherin while
tumor cells that have undergone EMT will be only YFP positive (Fig. 1b).
18. Centrifuge cells at 4
C at 300 RCF for 5 min and discard
supernatant.
19. Cells can now be processed for analysis to confirm EMT subtype by DNA, RNA, protein, etc. (see Note 7).
3.2 Isolation of YFPPositive Mesenchymal
Cells from Tissue
Culture by FACS (See
Note 8)
1. Aspirate media from 10 cm dish and wash the cells twice with
ice cold PBS.
2. Add 5 mL of enzyme-free, cell dissociation buffer and incubate
for 5–20 min or until cells have dissociated. Vigorously tap
every 5 min (see Note 9).
3. Neutralize media in 5 mL of staining buffer and centrifuge cells
at 4
C at 300 RCF for 5 min.
4. Discard the supernatant and add 500 μL staining buffer and
transfer cells to a microfuge tube.
5. Centrifuge cells at 4
C at 300 RCF for 5 min and discard
supernatant.
6. Continue with step 11 from Subheading 3.1.
320
Robert J. Norgard and Ben Z. Stanger
C at 300 RCF for 5 min and discard
supernatant. Wash pellet with 500 μL staining media. Repeat
for a total of three times.
13. Resuspend cells in 500 μL ice cold-staining buffer, add 5 μL
(1:100) APC donkey anti-rat secondary antibody and incubate
on ice for 15 min in the dark. Add 180 μL ice cold-staining
buffer to the 20 μL unstained negative control sample, and add
2 μL secondary antibody (1:100).
14. Centrifuge cells at 4
C at 300 RCF for 5 min and discard
supernatant. Wash with 500 μL staining media. Repeat for a
total of three times including negative control.
15. Resuspend cells in 1.5 mL sorting/staining buffer with 1.5 μL
Dapi (1:1000). Use an 18-gauge needle in a 3 mL syringe to
aspirate the cell solution. Remove the needle and then eject the
solution into a polystyrene round-bottom tubes with 35 μm
cell strainer cap. Depending on the type of FACS machine
being used, polypropylene round-bottom tubes may need to
be used. The filter cap can be switched onto these tubes or the
cells can be transferred into this type of tube. Keep the cells on
ice and dark until use.
16. Sort the cells in a FACs machine.
17. When processing using FACS, viable cells are impermeable to
DAPI, so DAPI positive cells are no longer viable and excluded
from the sort. It is important to use staining buffer without
phenol red, as this may interfere with fluorescent signal. Epithelial cells with be positive for YFP and E-cadherin while
tumor cells that have undergone EMT will be only YFP positive (Fig. 1b).
18. Centrifuge cells at 4
C at 300 RCF for 5 min and discard
supernatant.
19. Cells can now be processed for analysis to confirm EMT subtype by DNA, RNA, protein, etc. (see Note 7).
3.2 Isolation of YFPPositive Mesenchymal
Cells from Tissue
Culture by FACS (See
Note 8)
1. Aspirate media from 10 cm dish and wash the cells twice with
ice cold PBS.
2. Add 5 mL of enzyme-free, cell dissociation buffer and incubate
for 5–20 min or until cells have dissociated. Vigorously tap
every 5 min (see Note 9).
3. Neutralize media in 5 mL of staining buffer and centrifuge cells
at 4
C at 300 RCF for 5 min.
4. Discard the supernatant and add 500 μL staining buffer and
transfer cells to a microfuge tube.
5. Centrifuge cells at 4
C at 300 RCF for 5 min and discard
supernatant.
6. Continue with step 11 from Subheading 3.1.
320
Robert J. Norgard and Ben Z. Stanger