Esophageal Adenocarcinoma Methods and Protocols

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activated cell sorting (FACS) analysis. This two- step procedure

will reassure the quality of molecular analysis in these CTCs

including DNA amplification and immunological studies.

1. Prior to the CTC isolation, blood samples should be mixed
   with a one-half volume of cell buffer (CellSearch) solution for
   proper dilution.


2. The blood samples should initially treat with immunomagnetic
   particles coated with an EpCAM monoclonal antibody (mAb)
   (~50 μg/10 mL of the indirect conjugate and EpCAM mAb
   that is conjugated to phycoerythrin, ~5 μg/Ml per 10 mL).


3. Then, incubate the samples at room temperature for 15–30 min
   with appropriate mixing.


4. A magnetic separator will be needed to isolate the labeled par-
   ticles and the unbound blood cells will be aspirated out using
   a pipette.


5. The labeled/bound blood cells should later elute in cell buffer
   and will be subjected to the second round for magnetic separa-
   tion using the similar technique. This will help in increasing
   the sensitivity of purifying the bounded cells.


6. After obtaining the EpCAM-labeled cells following the second
   separation, a solution containing a nucleic acid dye (to stain
   the nucleated CTCs), and a leukocyte-specific CD45 mAb
   conjugated to peridinin-chlorophyll-protein-Cy5.5 will be
   added to the resuspended cells.


7. The samples will be incubated in dark for ~30 min prior to
   fluorescence assisted cell sorting (FACS) analysis.


8. As successful isolation of CTCs includes cells that are nucle-
   ated, EpCAM stained positive and CD45 stain negative cells.
   Leukocyte cells should be isolated and excluded from cells that
   are CD45 positive, EpCAM negative and nucleated.


9. Finally, the sorted cells will be eluted into a TE buffer (10 mM
   Tris, bring to pH 8.0 with hydrogen chloride and 1 mM EDTA)
   and stored in polymerase chain reaction tubes at − 80 °C.

4 Note

1. As the detection of CTCs is heavily dependent on the expres-
   sions of an epithelial cell adhesion molecule (EpCAM) and/or
   cytokeratin (CK), the varied CTC levels between adenocarci-
   noma and squamous cell carcinomas of the esophagus could
   also be attributed to the varied expression pattern of these
   markers. The most common definition of a CTC is an
   EpCAM+/CK+ and CD45− (lymphocyte antigen) cells in the
   peripheral blood with round or oval intracellular nucleus [ 24 ].

3.2 Antibody
Labeling

Vinod Gopalan and Alfred K. Lam