Esophageal Adenocarcinoma Methods and Protocols

173

4 Notes

1. Powdered media are highly hygroscopic and should be pro-
   tected from atmospheric moistures. Preparing a concentrated
   medium solution may form precipitation.


2. pH need to be reduced to 4.0 with 1 N HCl to dissolve the
   medium completely. After that, pH can be raised up to 7.2
   with 1 N NaOH prior to add sodium bicarbonate.


3. We noted that 10% FBS is good for the growth and mainte-
   nance of the esophageal adenocarcinoma cells (OE33).


4. The combined antibiotics, penicillin and streptomycin, were
   used to prevent the bacterial contamination of cultured OE33
   cells. We found that this concentration is enough to maintain
   and keep the cells free from bacterial contaminations.


5. Avoid repeated freeze thawing and warm up of trypsin.
   Multiple freezing, thawing, and warming may cause the
   reduced enzymatic activity of trypsin.


6. Hoechst 33342 dye should not be resolubilized in PBS, but
   dilute solutions of the dye may be used with PBS or other
   phosphate containing buffers. Refrigerated stock solution in
   water can be used at least for 6 months. Solutions of the
   Hoechst 33342 dye should be stored at 2–6 °C and protected
   from light. The dye is known as potential mutagen and should
   be handled with care. Disposal of the dye should be carried out
   safely in accordance with the proper regulations.


7. Make a stock solution of propidium iodide at 1 mg/mL
   (1.5 mM) in sterile distilled water and store at 2–6 °C. It is
   protected from light and can be used at least for 6 months.
   Propidium iodide is a potential mutagen, therefore use with
   care and dispose in accordance with the proper regulations.


8. We noted that addition of B27 (growth supplement) increased
   the spheres formation of sorted cells and attributed passages of
   spheres culture.


9. Animals should be 4–6 weeks old and allow at least 5 days
   acclimation period of mice arrival. Animal ethics from the
   proper authority must be approved and standard operating
   procedures must be followed in accordance with the approved
   guidelines.


10. Inhaled anesthesia (isoflurane) or local anesthesia (ketamine)
    make the implantation of cells to mice easier and less stressful.
    Isoflurane-induced anesthesia is performed by placing the
    mouse in a closed chamber/box with appropriate gas scaveng-
    ing attached. Once writhing reflex is lost, the animal may be
    removed from the induction chamber. The animal could be
    maintained on gas delivered via a fitted nose cone or endotra-

Cancer Stem Cells in Esophageal Adenocarcinoma