4 Notes
1. DNAse is critical to maintaining single cells suspensions. During the process, tumor and other cell types die and DNA is
released into the solution. This results in stickiness or clumping
of tumor cells.
2. MgCl 2 is necessary for the DNAse to work properly.
3. We are very careful when dissecting tumors. Anatomically (and
by use of fluorescent reporters, see Maddipati and Stanger [7]),
it can be determined whether a few distinct tumors exist within
a tumor mass. Therefore, we micro-dissect a tumor and typically cut it into halves or thirds depending on size. One half is
used for FACS or other molecular studies and the other half is
used for subsequent histological analysis.
4. As much as possible, steps that follow should be done in the
dark to preserve fluorescence of tumor cells. Typically, we wrap
our tubes or cover plates with aluminum foil.
5. This step is critical to quenching/diluting out collagenase
activity. The amount is variable but adding at least up to
30 mL is sufficient.
6. Inclusion of an isotype control primary antibody or secondary
antibody alone is best way to identify a true positive population. If performing this protocol with an unconjugated antibody (as in this protocol), use a secondary only control. If
performing this protocol with a conjugated antibody, use an
isotype control of the same type and fluorescence as the primary antibody.
7. A determination of EMT subtype can be done in several ways
(for more information, see Aiello et al. [6]). Briefly, RT-qPCR
can be used to distinguish EMT subtypes by measuring transcripts for epithelial genes (e.g., ECAD, EpCAM, Cldn7, etc.)
and mesenchymal genes (Vimentin, SPARC, FAP, etc.) comparing ECAD+ and ECAD- cells. After sorting tumor cells can
also be lysed and western blotting performed in a similar fashion to RT-qPCR.
8. This method is for isolating tumor cells that have undergone
EMT within a specific population in vitro or that have undergone EMT spontaneously or that have been induced to
undergo EMT by addition of a ligand such as TGF-β. A lineage
label is not necessary to separate tumor cells from non-tumor
cells in established cancer cell lines.
9. Trypsin is capable of cleaving E-cadherin and other cell surface
markers used to ascertain EMT status. Therefore, we use
enzyme-free cell dissociation buffer which yields better results
and safeguards from false negatives.
Isolation and Identification of EMT Subtypes
325
1. DNAse is critical to maintaining single cells suspensions. During the process, tumor and other cell types die and DNA is
released into the solution. This results in stickiness or clumping
of tumor cells.
2. MgCl 2 is necessary for the DNAse to work properly.
3. We are very careful when dissecting tumors. Anatomically (and
by use of fluorescent reporters, see Maddipati and Stanger [7]),
it can be determined whether a few distinct tumors exist within
a tumor mass. Therefore, we micro-dissect a tumor and typically cut it into halves or thirds depending on size. One half is
used for FACS or other molecular studies and the other half is
used for subsequent histological analysis.
4. As much as possible, steps that follow should be done in the
dark to preserve fluorescence of tumor cells. Typically, we wrap
our tubes or cover plates with aluminum foil.
5. This step is critical to quenching/diluting out collagenase
activity. The amount is variable but adding at least up to
30 mL is sufficient.
6. Inclusion of an isotype control primary antibody or secondary
antibody alone is best way to identify a true positive population. If performing this protocol with an unconjugated antibody (as in this protocol), use a secondary only control. If
performing this protocol with a conjugated antibody, use an
isotype control of the same type and fluorescence as the primary antibody.
7. A determination of EMT subtype can be done in several ways
(for more information, see Aiello et al. [6]). Briefly, RT-qPCR
can be used to distinguish EMT subtypes by measuring transcripts for epithelial genes (e.g., ECAD, EpCAM, Cldn7, etc.)
and mesenchymal genes (Vimentin, SPARC, FAP, etc.) comparing ECAD+ and ECAD- cells. After sorting tumor cells can
also be lysed and western blotting performed in a similar fashion to RT-qPCR.
8. This method is for isolating tumor cells that have undergone
EMT within a specific population in vitro or that have undergone EMT spontaneously or that have been induced to
undergo EMT by addition of a ligand such as TGF-β. A lineage
label is not necessary to separate tumor cells from non-tumor
cells in established cancer cell lines.
9. Trypsin is capable of cleaving E-cadherin and other cell surface
markers used to ascertain EMT status. Therefore, we use
enzyme-free cell dissociation buffer which yields better results
and safeguards from false negatives.
Isolation and Identification of EMT Subtypes
325