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- Select the gates for sorting into external collection tubes (see
Note 20). - Run the experimental sample tubes at 4 °C (see Note 21).
- Stop the sorting when desired number of cells were obtained
in the collection tubes and perform the post-sort analysis. - Collect the sorted cells in complete growth medium for in vitro
and in vivo experiments. - Count the fluorescence-activated cell sorting-isolated
SP-positive cells and adjust the cells number as 1000 cells/mL
in growth medium. - Add 100 μL of PBS to the first and last columns (column 1 and
12) of the 96-well ultralow attachment plate to minimize the
evaporation of tumor spheres formation medium. - Add 100 μL of tumor spheres formation medium into rest of
the wells. - Add the SP-positive cells in columns 2–11 at 200, 100, 50, 10,
5, and 1 cell into each well (see Note 22). - Seal the edges of the plate with lab-film to avoid evaporation of
medium. - Incubate the plate for 1–3 weeks at 37 °C with 5% CO 2 until
spheres formed. - Count the number of tumor spheres under phase-contrast
microscope using 40× magnifications and calculate the per-
centages of spheres formation capacity of the SP-positive cells. - Adjust the freshly fluorescence-activated cell sorting-isolated
SP-positive and SP-negative cells to a concentration of
1.0 × 107 cells/mL in cold PBS. Keep the cells always on ice. - Slowly pull up 100 μL of cells using 1 mL syringe fitted with
27- or 30-gauge needle (see Note 23). - Anesthetize the mice using isoflurane (see Note 10).
- Inject 1.0 × 106 cells into the flank of NOD/SCID mice. Pinch
the skin of the mouse between index finger and thumb and
pull the skin away from the body of the mouse. - Inject the cells slowly and evenly into the created pouch.
- Monitor the tumor growth by palpation and sacrifice the ani-
mals when largest subcutaneous tumors reach ~50–60 mm^3 in
diameter. - Measure tumor diameter with digital caliper and calculate
tumor volume in mm^3 using the formula:
Volume = (width)^2 × length/2.
3.3 In Vitro Tumor
Spheres Formation
Assay
3.4 Xenotrans
plantation
Farhadul Islam et al.