Esophageal Adenocarcinoma Methods and Protocols

(sharon) #1

172



  1. Select the gates for sorting into external collection tubes (see
    Note 20).

  2. Run the experimental sample tubes at 4 °C (see Note 21).

  3. Stop the sorting when desired number of cells were obtained
    in the collection tubes and perform the post-sort analysis.

  4. Collect the sorted cells in complete growth medium for in vitro
    and in vivo experiments.

  5. Count the fluorescence-activated cell sorting-isolated
    SP-positive cells and adjust the cells number as 1000 cells/mL
    in growth medium.

  6. 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.

  7. Add 100 μL of tumor spheres formation medium into rest of
    the wells.

  8. Add the SP-positive cells in columns 2–11 at 200, 100, 50, 10,
    5, and 1 cell into each well (see Note 22).

  9. Seal the edges of the plate with lab-film to avoid evaporation of
    medium.

  10. Incubate the plate for 1–3 weeks at 37 °C with 5% CO 2 until
    spheres formed.

  11. 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.

  12. 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.

  13. Slowly pull up 100 μL of cells using 1 mL syringe fitted with
    27- or 30-gauge needle (see Note 23).

  14. Anesthetize the mice using isoflurane (see Note 10).

  15. 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.

  16. Inject the cells slowly and evenly into the created pouch.

  17. Monitor the tumor growth by palpation and sacrifice the ani-
    mals when largest subcutaneous tumors reach ~50–60 mm^3 in
    diameter.

  18. 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.
Free download pdf