169media to the water (see Note 1). Stir until it completely dis-
solves and do not heat the media (see Note 2). Weigh 2.0 g of
sodium bicarbonate and add to the media. Stir for complete
dissolving and adjust the pH with 1 N HCl (hydrogen chlo-
ride) and NaOH (sodium hydroxide). Add water up to mark
1 L and sterilize with 0.22 μm membrane filter. Aseptically
transfer media into sterile container and store at 4 °C.- 10% fetal bovine serum (FBS) in sterile media (see Note 3).
Store at 4 °C. - 1% mixture of penicillin and streptomycin antibiotic in sterile
media and store at 4 °C (see Note 4). - Cell washing buffer: Phosphate buffer saline (PBS), pH 7.4.
Add 8.0 g NaCl, 0.2 g KCl, 1.42 g Na 2 HPO 4 , and 0.24 g
KH 2 PO 4 in glass beaker. Dissolve all the salts in 800 mL water
and adjust the pH with 1 N HCl. Add water to a total volume
of 1 L. Sterilize and store at 4 °C. - Cell dissociation solution: 0.25% Trypsin-
ethylenediaminetetraacetic acid (EDTA), pH 7.2–8. Stored at
− 20 °C (see Note 5). - Cell culture flasks: Polystyrene 25 cm^2 and 75 cm^2 flasks.
- Falcon 15 and 50 mL conical centrifuge tubes.
- Microscope and centrifuge machines.
- 80% ethanol in water (v/v).
- Cell staining dye: 5 μg/mL Hoechst 33342 in water (see Note 6).
- Dead cell dye: 10 μg/mL propidium iodide (PI) in water (see
Note 7). - Wash buffer: Cold PBS at pH 7.2.
- ATP-binding cassette- (ABC) transporter inhibitor: 50 μM
verapamil or 10 μM fumitremorgin C. - Centrifuge tubes—1.5 mL.
- Vortex mixer.
- Flow cytometer: Equipped with
(a) Excitation wavelength 325 nm or 351–364 nm.
(b) Detection filters—450/20 nm bandpass (BP) filter,
675 nm longpass (LP) filter, and 610 nm shortpass
dichroic mirror. - Cell counter/Hemocytometer.
- 0.4% trypan blue solution in PBS, pH 7.2–7.3.
- Polystyrene tubes—5 mL.
- Collection tube: 15 mL falcon tube.
2.2 Cell Staining
and Sorting
Cancer Stem Cells in Esophageal Adenocarcinoma