Esophageal Adenocarcinoma Methods and Protocols

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  1. Cell culture plates: 6-well plates, 96-well ultralow attachment
    plates.

  2. Cell culture media: RPMI1640 (see Subheading 2.1).

  3. Tumor spheres formation medium: RPMI1640, 50× B27,
    20 ng/mL epidermal growth factor (EGF), 10 ng/mL basic
    fibroblast growth factor (FGF), 5 μg/mL insulin. Add 10 μg
    EGF, 5 μg FGF, 2.5 μg insulin, and 0.5 mL B27 supplement
    to 500 mL of RPMI1640 media (see Note 8). Store at 4 °C.

  4. Cell culture medium: RPMI1640 (see Subheading 2.1).

  5. Cancer cells: SP-positive, SP-negative.

  6. Animal: Non-obese diabetic/severe combined immunodefi-
    ciency (NOD/SCID) (see Note 9).

  7. Anesthetic agents: 1–3% isoflurane, 80–100 mg/kg ketamine
    (intraperitoneal) (see Note 10).

  8. Syringe: 1 mL with 27- or 30-gauge needle.

  9. Digital caliper.


3 Methods


Unless otherwise mentioned, perform all the experiments at room
temperature.


  1. Take the cryovial containing esophageal adenocarcinoma cells
    (OE33) from liquid nitrogen facility and transfer them into a
    37 °C water bath.

  2. Quickly thaw the cells by gently swirling the vial in water bath
    at 37 °C (see Note 11).

  3. Transfer the vial into a biosafety cabinet and wipe out site of
    the vial with 80% ethanol.

  4. Add pre-warmed complete growth media (2 mL) in a 15 mL
    centrifuge tube and add the thaw cells dropwise into the tube.

  5. Centrifuge the cell suspension at approximately 400 g for
    3–5 min.

  6. Check the cell pellet after centrifugation and aseptically decant
    the supernatant without disrupting the cells pellet.

  7. Add 2 mL PBS and resuspend the cells pellet with slow pipetting.

  8. Centrifuge the suspension at 400 × g for 3–5 min and discard
    the supernatant.

  9. Resuspend the cell pellets in complete growth media (contain-
    ing FBS) and transfer them into an appropriate cell culture
    flask containing recommended growth media and incubate in
    the CO 2 incubator at appropriate conditions (see Note 12).


2.3 In Vitro
Functional Assay


2.4 In Vivo
Transplantation


3.1 Cell Culture


Farhadul Islam et al.
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