Store at 4℃ away from light for 3 months.

1. Primary cell culture of esophageal cancer

The suspension of small samples of esophageal cancer cells isolated for the first time is usually implanted directly into the 12-well plate without counting. During esophageal cancer surgery, the samples were counted through a 70-micron screen and inoculated into a suitable culture vessel according to the number of cells in Table 1 of the instructions. NIH-3T3 cells irradiated by gamma rays were added (the number of trophoblast cells added is shown in Table 1 and Table 2 of the instructions). The primary cells were not moved for 2-3 days after the initial inoculation (which is conducive to cell adhesion). During the culture process, if the medium color turns yellow but the cells are not overgrown, half of the solution can be replaced. When the cells are not overgrown but the NIH-3T3 cells are insufficient, the NIH-3T3 cells irradiated by gamma rays should be properly supplemented (generally half of the initial number).

2. Primary cell passage of esophageal cancer

The cells were observed to form clones under microscope, and the confluence was 80~90%. The cells were removed from the incubator, the old culture medium was discarded, 0.25% pancreatic enzyme was washed for 30 seconds and sucked up, and an appropriate amount of 0.05% pancreatic enzyme was added, incubated at 37℃ for 5 minutes, and the cell digestion was observed under a microscope. When the cells became round and began to fall off, the digestion was terminated with a stop medium, and the cells were transferred to a centrifuge tube and centrifuged at 1500 rpm for 3 min. The supernatant was abandoned, the cells were re-suspended with 1-2 mL of the corresponding primary cell medium, the live cells were counted, the cell suspension was inoculated on the culture bottle/plate according to the appropriate cell density, and the NIH-3T3 cells irradiated by gamma rays were added (see Table 1 in the instructions for different sizes of the bottom area of the culture bottle/plate, the number of primary cells inoculated, and the number of trophoblast cells added). The primary cell medium was supplemented to the required volume, gently shaken and mixed, and cultured in an incubator at 37 ℃ and 5% CO2 after surface disinfection. Do the same for other steps.