Cell Proliferation Assay

　　1 Cell recovery and culture

　　1.1 According to customer needs, resuscitate relevant cells. Recovery and culture of each cell was performed according to relevant SOPs. After the cells are recovered and cultured for 3-5 days, the cell viability rate reaches >95% before they can be used for cell proliferation experiments. If the customer's experiment is to screen multiple cell lines for the same compound, a 25 cm2 culture flask can be used for cell culture; if the same cell line is used to screen multiple compounds, a 75 cm2 cell culture flask can be used. nourish.

　　2 cell seeding

　　2.1 After cells are collected and digested with Trypsin, count the cells. Then seed 96-well white microplate at 4000 cells/100 μL per well. The layout of the microplate during inoculation is shown in Figure 1. When inoculating, the cells need to be mixed evenly, poured into the sample tank, and then added to the microplate using a multi-channel pipette. Let the microplate stand in the biosafety cabinet for 20 minutes and then transfer to the CO2 incubator.

　　Notice:

　　1) Just add PBS to the peripheral wells of the microplate to reduce the edge effect;

　　2) After adding cells to the microwell plate, keep the microwell plate as still as possible to avoid uneven distribution of cells;

　　3) Be as gentle as possible when opening and closing the CO2 incubator to avoid shaking the cells.

　　3 Compound solution preparation and sample addition processing

　　3.1 Preparation of 10 mM DMSO stock solution

　　Weigh the appropriate amount of compound to prepare a 10 mL DMSO stock solution. After preparation, pay attention to observe whether there is any precipitation. If there is any precipitation, it can be dissolved by shaking or heating with a hair dryer. If the compound's solubility in DMSO is less than 10 mM, further dilution of the compound to 5mM may be considered. The prepared storage solution is aliquoted as needed and stored at -20°C.

　　Note: DMSO needs to be at least HPLC grade or biological experiment grade, and avoid opening it repeatedly. DMSO is hygroscopic, and repeated opening may cause an increase in water molecules in the DMSO, reducing the stability of the compound.

　　3.2 Preparation of compound gradient dilution solution (Serial Dilution in DMSO) (taking the starting concentration as 10 μM as an example)

　　3.2.1 Take a clean and sterile V-shaped bottom or U-shaped bottom 96-well microplate and place it in the biological safety cabinet;

　　3.2.2 First add 8 μL DMSO to well B2 and 10 μL DMSO to wells B3-B11;

　　3.2.3 Then add 12 μL of 10 mM compound stock solution into well B2, and mix by pipetting up and down 10 times; at this time

　　The concentration of compound in well B2 was 6 mM, 100% DMSO;

　　3.2.4 If there are multiple compounds, follow the above two steps to prepare the relevant compound solutions in wells C2-G2 respectively;

　　3.2.5 Take 5 μL compound solution from well B2-G2, transfer it to B3-G3, pipe up and down 10 times to mix;

　　3.2.6 Then dilute sequentially until diluted to B10-G10; the layout of the compound microplate after dilution is shown in Figure 2 below.

　　3.3 Preparation of 6X compound solution in culture media

　　3.3.1 Take a clean and sterile V-shaped bottom or U-shaped bottom 96-well microplate and place it in the biological safety cabinet;

　　3.3.2 Add 99μL of cell culture medium to each well of B2-G11;

　　3.3.3 Use a 12-channel pipette to add the compound solution prepared in step 3.2 to the corresponding well, adding 1 μL to each well;

　　3.3.4 Add 100μL PBS to each surrounding well.

　　3.4 Compound treatment of cells

　　3.4.1 Take out the cell culture plate inoculated the day before from the incubator;

　　3.4.2 Use a 12-channel pipette to pipet and mix the compound solution prepared in step 3.3;

　　3.4.3 Take 20 μL of the compound solution from each well and add it to the corresponding cell culture well. When adding the sample, select the lowest pipetting speed, adhere to the wall and avoid contact with the bottom of the culture plate;

　　3.4.4 Gently shake the cell culture plate to mix the compound solution, then place the cell culture plate back into the incubator and continue culturing for 72 hours.