Esophageal cancer is a malignant lesion formed by abnormal proliferation of esophageal squamous or glandular epithelium. Its development generally passes through the stages of epithelial atypical hyperplasia, carcinoma in situ and invasive carcinoma.
Operation method
Cell Culture Technology
Materials and Instruments
Human Esophageal Cancer Cell Line Move I. Preparation of materials For more product details, please visit Aladdin Scientific website.
Trypsin RNAase Propidium iodide Tetramethyl azole salt Calf serum DMEM DMSO K2HPO4 Na2HPO4 NaCI
Sterile Sorting Tubes 400 Mesh Cell Sieve Water Bath Centrifuge Enzyme Labeler Electrophoresis Microscope PCR Instrument
1. Penicillin storage solution
(1) Fully dissolve 800,000 units of dry powder from the penicillin vial in 8 ml of deionized water.
(2) Dispense into 1 ml/tube and store at -20°C.
2. Streptomycin storage solution
(1) Fully dissolve 1 mg of the dry powder from the vial of streptomycin in 10 ml of deionized water.
(2) Dispense into 1 ml/tube and store at -20°C.
3. RPMI 1640 medium
(1) Fill a large triangular flask with 800 ml of deionized water.
(2) Add 1 sachet of RPMI 1640 dry powder to make bauhinia and mix on a blender.
(3) Add 2.0 g Na2HCO3, 1 ml 100,000unit/ml penicillin storage solution.
(4) 1 ml fully dissolved, adjust the pH to 7.4, deionized water was fixed to 1000 ml.
(5) Filtered by 0.22 um pore size filter membrane under sterile conditions and stored at 4℃ in separate units.
4. UltraMEM culture solution
(1) 500 ml UltraMEM with sterile 10 ng/ml bFGF, 10 ng/ml EGF, 50 U/ml penicillin and 50 ug/ml streptomycin, stored at 4°C.
(2) Since the completely serum-free state is unfavorable for cell growth, we added 5% FBS to this culture medium to maintain FBS at a low level.
5. 1 x PBS (pH 7.4)
(1) Dissolve 1.15 g Na2HPO4, 0.2 g K2HPO4, 8.0 g NaCl, and 0.2 g KCl in 800 ml of deionized water.
(2) Adjust the pH to 7.4 with 1 M HC1 and add water to make a volume of 1000 ml.
6. EGF: Dissolve 100 ug of EGF in ampoule with 1 ml of sterile water, divide it into 10 tubes, take 1 tube and dilute it 100 times and divide it into small portions to be used as the working solution, and the remaining 9 tubes will be used as the storage solution, and all of them will be placed at -20℃ for storage.
7. bFGF
(1) Fully dissolve 10 ug of bFGF within the ampulla with 1 ml Tris buffer
(2) Dispense into 10 tubes, dilute each tube 10-fold with PBS and then dispense into small portions as working solution and store at -20°C.
8. 0.25% trypsin
(1) Weigh 0.25 g of trypsin and dissolve in 1x PBS 100 ml.
(2) Filter through 0.22 um pore size membrane under sterile conditions and store at 4°C.
9. 5 mg/ml tetramethyl azole salt (MTT)
(1) 0.5 g of MTT powder was added to 100 ml of PBS bath solution and fully dissolved.
(2) Filtered through 0.22 um pore size membrane under sterile conditions and stored at -20°C in separate units.
10. Cell freezing solution: DMSO:FBS=1:9, stored at -20°C.
11. Hoechst 33342
(1) Dissolve 10 mg of Hoechst 33342 powder in 1 ml of sterile water as a storage solution.
(2) Remove 10 ul and dilute to 40 ul as working solution, both of which are stored at -20°C, protected from light.
12. Propidium iodide (PI)
(1) Mix 25 mg PI powder in 1 x PBS buffer 250 ml avoiding light until fully dissolved.
(2) Obtain the final concentration of l00 ug/ml of storage solution, filtered and dispensed, and stored at 4 ℃ away from light.
13. 10 mg/ml RNAase
(1) Tryptic RNAase was dissolved in solvent [10 mM Tris-HCl (pH 7.5), 15 mM NaCl] at a concentration of 10 mg/ml.
(2) Place at 100°C for 15 min, slowly cool to room temperature and dispense as 200 ul/branch and store at -20°C.
14. Verapamil formulation
(1) Prepare stock solution with DMSO at a concentration of 50 mg/ml.
(2) The concentration of the application solution is 100 umol/ml from the literature.
(3) Therefore, the concentration of 100 umol/ml can be obtained by diluting the stock solution 50 times.
(4) In the experiment, adding 10 uL of the stock solution to each ml of cell suspension can achieve the concentration needed to block the off-channel.
Experimental Procedure
1. Flow cytometry sorting method:
(1) 0.25% trypsin digested nasopharyngeal carcinoma cells in logarithmic growth phase, 1200 rpm/min, centrifuged for 5 min, discarded the supernatant and added 5 ml PBS to resuspend the cells, centrifuged and washed twice.
(2) Cells were resuspended in ice pre-cooled complete culture medium containing 2% FBS RPMI 1640, adjusted to a concentration of 1 × 106 cells/ml, and incubated for 10 min at 37°C in an incubator.
(3) Add fluorescent dye Hoechst 33342 at a concentration of 7.5 ug/ul to a final concentration of 5 ug/ml, avoid light, and incubate for 90 min at 37°C in an incubator, mixing intermittently to avoid cell sinking.
(4) Centrifuge at low temperature, 4℃, 1200 rpm/min, 5 min, discard the supernatant.
(5) 2 ml PBS resuspended at 1200 rpm/min for 5 min.
(6) Resuspend the cells with 2~4 ml RPMI1640 culture medium and sieve the cells with a 400 mesh autoclaved cell sieve to remove adherent cell mass.
(7) Add PI at a concentration of 50 ug/ml to reach a final concentration of 1~2 ug/ml 5 minutes before the flow cytometer, and label the cells by avoiding light.
(8) On-machine analysis or sorting.
2. Crystalline violet staining procedure:
(1) Wash each well twice with PBS to remove protein components from the culture medium.
(2) Add 1.5 ml of 100% ethanol per well and fix for 15 minutes at room temperature.
(3) Wash twice with PBS and add 1 ml of 4% crystalline violet dye to each well. Act for 15 minutes at room temperature.
(4) Wash away excess dye slowly under running water, control the water on absorbent paper, count the number of clones with cell numbers greater than 50 and calculate the CFE.
3. Research on multidirectional differentiation and self-renewal capacity:
(1) SP and NSP cells were inoculated separately in culture flasks after sorting.
(2) After culturing for 2 weeks to reach the number of cells for reanalysis, the cells were collected for Hoechst 33342 staining to analyze the high percentage of SP cells.''
4. Total cellular RNA extraction:
(1) Cellular RNA was extracted by Trizol Reagent. SP and NSP cells with good growth status were taken, culture medium was discarded, cells were digested with 0.25% trypsin, washed twice with sterile PBS, 1 ml Trizol was added, and the cells were lysed with the cells and then transferred into EP tubes, and left to stand for 5 min at room temperature.
(2) Add 0.3 ml of chloroform, invert and mix the EP tube for 15 sec, let it stand at room temperature for 3 min, centrifuge at 12 000 g at 4°C for 10 min, and slowly aspirate the supernatant into another EP tube.
(3) Add 0.75 ml isopropanol, mix well, let stand at room temperature for 10 min, centrifuge at 12 000 g for 10 min at 4°C, and discard the supernatant.
(4) Add 75% ethanol (prepared with DEPC-treated deionized water), mix, and centrifuge at 4°C, 7,500 g for 5 min; discard supernatant.
(5) Dry at room temperature, add 20 ul of DEPC-treated deionized water, and melt in a water bath at 55℃~60℃ for 10 min, then dilute 1:25 to detect the 260:280 ratio and RNA concentration, divide, and store in a refrigerator at -80℃ for spare use.
5. In vivo tumor formation test in mice
(1) Pre-experiments were first done with unsorted EC-9706 cells to determine the number of inoculated cells, and the number of selected plots were 1×106, 1×105 and 1×104, respectively, and the tumor formation was observed after four weeks.
(2) The SP and NSP cells obtained from the sorting in the morning of the same day were accurately counted and resuspended in RPMI-1640 culture medium, and the cells were inoculated in 6 groups with an inoculation volume of 100 ul.
(3) were stored at 4°C in the animal room and injected into the axilla of NOD/SCID mice separately, 3 mice per dose, for a total of 24 mice for SP and NSP cells.
(4) Feed for 4 to 9 weeks and observe tumor growth twice a week.
(5) Measure the tumor size with vernier calipers before execution, execute the mice by neck-breaking method, and take photos to remove the tumor tissues.
(6) The clipped portion was immersed and fixed with 10% formalin, and the sections were subjected to HE staining to determine the pathological type.
6. Western Blot analysis of target proteins:
(1) Extraction of total cellular protein
① Prepare the ice box and do the following operations on ice as much as possible.
② Wash away the residual culture solution in the cell culture dish with ice pre-cooled PBS.
③ Add 1 ml of PBS, scrape the cells off with a cell scraper and collect them in an EP tube and centrifuge at 1500 rpm/min for 5 min or 1800 rpm/min for 3 min.
④ Slowly aspirate the supernatant, add 1 ml of cell lysis solution to the lower cell precipitate to lysed the cells, mix well and leave on ice for at least 45 min, then centrifuge for 5 min at 4°C, 12,000 m/min on a low-temperature ultracentrifuge.
⑤ Aspirate the supernatant, dispense and assay the protein concentration.
(2) Plotting of standard curves and determination of sample protein concentration
① Dilution of BSA protein standard: The stock solution with a concentration of 2000 ug/ml was gradually diluted to obtain a gradient of standards with concentrations of 1500 ug/m, 1000 ug/m, 750 ug/m, 500 ug/m, 250 ug/m, 125 ug/m and 25 ug/ml, respectively.
② Preparation of working solution: mix 50 volumes of Liquid A with 1 volume of Liquid B according to the instructions.
③ Take 5 ul of standard or sample, add 100 ul of working solution and mix for 30 seconds at room temperature.
After incubation at 37℃ for 30 min and reduced to room temperature, the OD value was measured at 570 nm, the standard curve was plotted and the concentration of the protein sample was calculated according to the dilution.7. Immunofluorescence assay to analyze the expression of cellular protein molecules:
(1) Carefully put the treated coverslips into the six-well plate plate with tweezers, add the droplets of SP and NSP cells received from sorting to the sheet respectively, use surface tension to control the droplets within the sheet, let it stand for 20~30 min, gently replenish the culture solution after the cells are attached to the wall (be careful not to make the sheet float), and put it into incubator to be cultured overnight.
(2) On the next day, the culture medium in the wells was discarded, washed three times with PBS, 10% formaldehyde was added, and fixed at 4°C for 30 min.
(3) Wash 3 times with PBS to remove paraformaldehyde, and add 1 ml of 5% bovine serum albumin to each well and incubate at room temperature for 2 h to block non-specific IgG binding.
(4) Wash twice with PBS, add appropriate amounts of CD44 and CK18 primary antibodies, and place on a shaker at 4°C overnight.
(5) Wash 3 times with PBS-T (PBS containing 0.02% Tween 20), add appropriate amount of fluorescent secondary antibody, place in a dark box, and place on a shaker for 2 hours at room temperature for binding.
(6) Wash 3 times with PBS-T, fix the coverslips on the slides with sealing solution containing 0.5 mg/ml DAPI, and store in a dark box protected from light.
(7) Observe the fluorescence intensity of the cells under an Olympus orthogonal fluorescence microscope, record and take photographs.