15. Apply cell suspension onto the column and collect flow
through into a 15 mL conical tube containing unlabeled or
poorly labeled cells (these will be used for the LD column).
16. Wash column 3 Â 3 mL with MACS buffer and collect flow
through as in step 15.
17. Remove the column from the separator and place it on a
suitable collection tube (15 mL conical will suffice).
18. Apply 5 mL MACS column and immediately flush out magnetically labeled cells with the plunger into the column. This is
the epithelial ECAD positive or ECAD-high population.
19. Place a LD column in a magnetic field of a suitable MACS
separator (QuadroMACS separator if multiple columns are
being used simultaneously).
20. Prepare a LD column by rinsing 2 mL of MACS buffer on top
of the column. Discard the effluent and change collection tube.
21. Apply the effluent from steps 15 through 16 and collect the
flow through.
22. Wash LD column with 2 Â 1 mL MACS buffer and collect flow
through (see Note 12). This is the mesenchymal, EMT ECAD
negative or ECAD-low population. These populations can
then be confirmed by flow cytometry for purity (Fig. 2b).
23. There are several advantages and disadvantages with FACS
versus MACS that are discussed in Table 1.
3.4 Identification of
Partial EMT Tumor
Cells by Surface/
Intracellular Flow for
Epithelial Proteins
Partial EMT tumor cells exhibit both epithelial and mesenchymal
characteristics. In recent work, we found that some tumor cells
re-localize epithelial proteins from the cell surface to the intracellular space. Therefore, it is possible using a dual antibody staining to
identify whether the protein has moved from the cell surface to an
intracellular compartment.
1. Prepare single cell suspensions from primary tumors (see steps
1–10 from Subheading 3.1) or tissue culture (see steps 1–5
from Subheading 3.2).
2. Resuspend cells in 300 μL ice cold-staining buffer and transfer
to the well of a 96-well dish. Save a small aliquot of cells or split
sample in half prior to adding the primary as an unstained
negative control. This will receive secondary antibody in the
following steps (see Note 6).
3. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant by inverting the plate with a quick twitch.
4. Resuspend in 100 μL staining buffer with 1:250 rat anti-ECAD
primary antibody and incubate on ice for 15 min.
5. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant by inverting the plate with a quick twitch.
Isolation and Identification of EMT Subtypes
323
through into a 15 mL conical tube containing unlabeled or
poorly labeled cells (these will be used for the LD column).
16. Wash column 3 Â 3 mL with MACS buffer and collect flow
through as in step 15.
17. Remove the column from the separator and place it on a
suitable collection tube (15 mL conical will suffice).
18. Apply 5 mL MACS column and immediately flush out magnetically labeled cells with the plunger into the column. This is
the epithelial ECAD positive or ECAD-high population.
19. Place a LD column in a magnetic field of a suitable MACS
separator (QuadroMACS separator if multiple columns are
being used simultaneously).
20. Prepare a LD column by rinsing 2 mL of MACS buffer on top
of the column. Discard the effluent and change collection tube.
21. Apply the effluent from steps 15 through 16 and collect the
flow through.
22. Wash LD column with 2 Â 1 mL MACS buffer and collect flow
through (see Note 12). This is the mesenchymal, EMT ECAD
negative or ECAD-low population. These populations can
then be confirmed by flow cytometry for purity (Fig. 2b).
23. There are several advantages and disadvantages with FACS
versus MACS that are discussed in Table 1.
3.4 Identification of
Partial EMT Tumor
Cells by Surface/
Intracellular Flow for
Epithelial Proteins
Partial EMT tumor cells exhibit both epithelial and mesenchymal
characteristics. In recent work, we found that some tumor cells
re-localize epithelial proteins from the cell surface to the intracellular space. Therefore, it is possible using a dual antibody staining to
identify whether the protein has moved from the cell surface to an
intracellular compartment.
1. Prepare single cell suspensions from primary tumors (see steps
1–10 from Subheading 3.1) or tissue culture (see steps 1–5
from Subheading 3.2).
2. Resuspend cells in 300 μL ice cold-staining buffer and transfer
to the well of a 96-well dish. Save a small aliquot of cells or split
sample in half prior to adding the primary as an unstained
negative control. This will receive secondary antibody in the
following steps (see Note 6).
3. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant by inverting the plate with a quick twitch.
4. Resuspend in 100 μL staining buffer with 1:250 rat anti-ECAD
primary antibody and incubate on ice for 15 min.
5. Centrifuge cells at 4
C at 300 RCF for 1 min and remove
supernatant by inverting the plate with a quick twitch.
Isolation and Identification of EMT Subtypes
323