171- Grow the esophageal adenocarcinoma cells up to 70–80% con-
fluency and harvest the cells after trypsinization. - Take the flask (s) inside biosafety cabinet and discard the
media, rinse with 1× PBS. - Vacuum off the PBS and add 0.25% trypsin-EDTA to the flask
and incubate for 5–10 min or until disassociation of cells (see
Note 13). - After completing the detachment of cells from the flask, add
growth media to neutralize EDTA. - Rinse the flask and pipette off cells to ensure maximum collec-
tion of cells. - Centrifuge for 3–5 min at 400 × g, discard the media, and wash
the cells with PBS. - Resuspend cells in cold PBS and count the cells with a cell
counter or hemocytometer using trypan blue (see Note 14). - Readjust the esophageal adenocarcinoma cell number to
1 × 107 cells/mL in cold PBS (see Note 15). - Label tubes as “Inhibitor,” “SP-stained,” and “Unstained” (see
Note 16). - Take 100 μL of prepared cell suspension (equal to one million
cells) to each tube. - Add 10 μL of verapamil to the “Inhibitor” tube.
- Add 10 μL of Hoechst 33342 stain to the “SP-stained” and
“Inhibitor” label tubes. - Vortex gently for few seconds and incubate for 15–30 min in a
covered ice bucket. - Add 1–2 mL cold PBS to wash off excess staining and centri-
fuge for 5 min at 800 × g. - Aspirate the supernatant without disturbing the cells pellet.
- Resuspend the cells in 500 μL cold PBS and add 5 μL propid-
ium iodide stock solution to “SP-stained” and “Inhibitor”
label tubes. - Incubate for 15–30 min on ice and keep in dark.
- Wash off excess propidium iodide with cold PBS and resus-
pend cells in 500 μL cold PBS with 1% bovine serum albumin
(see Note 17). - Mix the cell suspension with pipette up and down several
times. Then, transfer to the fluorescence-activated cell sorting
tube for analysis and sorting. - Set up and optimize the cell sorter (see Note 18).
- Perform compensation and set gates for controls and stained
samples (see Note 19).
3.2 Cell Staining
and Sorting
Cancer Stem Cells in Esophageal Adenocarcinoma