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tremorgin C. Cells treated with inhibitor are used to set gate
and therefore can discriminate SP population from non-SP
cells. Unstained cell group is needed for the initial gate setting
for the cell type analysis.
- Cells can be kept in refrigerator (for up to 1 week) for later
analysis with flow cytometer. The sorting buffer (PBS with 1%
FBS) is used to avoid autofluorescence. The high pressure dur-
ing cell sorting can cause sorting buffer to become basic. Add
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES)
(25 mM) to maintain the stable pH at 7.0–8.0.
- Flow cytometer setup varies depending on the manufacturer
and is performed by trained personnel. For this, first select the
nozzle and sterilize the instrument. Perform the quality con-
trol with beads to check the lasers and functionality of the
instrument. Then install the 450/20 BP filter for Hoechst
Blue, the 675 LP filter for Hoechst Red detection, and the 610
shortpass dichroic mirror for separation of Blue and Red sig-
nals. Propidium iodide fluorescence generated from ultraviolet
excitation will also be captured by the 675 LP filter. High fluo-
rescence signals produced by propidium iodide- positive dead
cells need to be discriminated from Hoechst Red-positive sig-
nals produced by live cells.
- Compensation is essential to remove the spectral overlap
between the detectors. Based on current literature, compensa-
tion is not absolute. It is affected by intensity of signals pro-
duced by certain markers and by the signals of cells that have
low autofluorescence, which led to the poor resolution between
dim and negative populations. To create appropriate gating for
the isolation of SP cells, first plot Forward Scatter (FSC, related
to cell size) and Side Scatter (SSC, related to cell granularity)
to discriminate cells from debris. A dot plot of Hoechst Blue
versus height is created to differentiate single cells from cell
aggregates and doublets. Finally, Hoechst Blue versus Hoechst
Red plot is created for SP isolation. ABC-transporter inhibitor
verapamil treated cells are used to establish the SP gate. Run
the sample and adjust the FSC versus SSC plot by changing
voltages of both parameters until all cells are captured on the
dot plot. Adjust the voltages of Hoechst Blue and Red param-
eters until the cells stained brightly for Hoechst 33342 are vis-
ible on the dot plot. Increase the voltages until the cells stained
weakly for Hoechst 33342 are visible on the lower left side of
the plot. SP cells constitute a discrete cells population on the
left side of the plot.
- In two sorting (SP-positive and SP-negative), 15 mL falcon can
be used. To prevent sorting cells sticking to the side of the tube,
coat the tubes with bovine serum albumin. Fill the tubes with
10% bovine serum albumin and incubate at 4 °C overnight.
Cancer Stem Cells in Esophageal Adenocarcinoma
