Experimental procedure

Suspension cells:

1) Collect the logarithmic phase cells, adjust the concentration of the cell suspension to 1 × 106 / ml, and add ① supplemented 1640 (serum-free) medium 40ul in sequence; / ml, pre-test to find the best dilution, 1: 10-1: 20); ③10ul of the test substance is required; ④50ul of cell suspension (ie 5 × 104cell / well), add 100ul to 96-well plate (edge Fill the hole with sterile water). Set a control for each plate (add 100? (Storage solution 100 1640).

2) Incubate at 37 ° C, 5% CO2 for 16-48 hours, and observe under an inverted microscope.

3) Add 10 ul MTT solution (5 mg / ml, or 0.5% MTT) to each well and continue culturing for 4 h. (WST-1 is recommended for suspension cells. Step 4 can be skipped after 4 hours of culture.) Direct enzyme-linked immunoassay detector OD570nm (630nm calibration) to measure the absorbance of each well)

4) Centrifuge (1000 rpm x 10 min), carefully aspirate the supernatant, add 100 ul dimethyl sulfoxide to each well, and shake on a shaker at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at OD570nm (630nm calibration) of enzyme-linked immunoassay.

5) Set up zero-adjusting wells (medium, MTT, dimethyl sulfoxide) and control wells (cells, drug dissolution medium with the same concentration, culture solution, MTT, dimethyl sulfoxide), set 3 times for each group hole.

Adherent cells:

1. Collect the logarithmic phase cells, adjust the concentration of the cell suspension, add 100ul to each well, and plate to adjust the density of the cells to be tested to 1000-10000 wells (the edge wells are filled with sterile PBS).

Incubate with 2.5% CO2 at 37 ° C until the cell monolayer is covered with the bottom of the well (96-well flat bottom plate). Add a concentration gradient of the drug. In principle, the drug can be added after the cell adheres to the wall, or two hours or half a day, but We often lay the plate in the afternoon of the previous day and add medicine in the morning of the next day. Generally, 5-7 gradients, 100ul per hole, 3-5 multiple holes are recommended. It is recommended to set 5, otherwise it will be difficult to reflect the real situation

Incubate with 3.5% CO2 at 37 ℃ for 16-48 hours, observe under an inverted microscope.

4. Add 20ul MTT solution (5mg / ml, or 0.5% MTT) to each well and continue culturing for 4h. If the drug can react with MTT, you can centrifuge and discard the culture solution. After carefully washing with PBS 2-3 times, add the culture solution containing MTT.

5. Add 150ul of dimethyl sulfoxide to each well, and shake on a shaker at low speed for 10min to fully dissolve the crystals. The absorbance of each well was measured at OD490nm of the enzyme-linked immunoassay detector.

6. Terminate the culture and carefully aspirate the culture medium from the well.

7. Set up zero-adjusting wells at the same time (medium, MTT, dimethyl sulfoxide), control wells (cells, drug dissolution media of the same concentration, culture solution, MTT, dimethyl sulfoxide)

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