3D Cell Culture

　　1. Preparation before experiment

　　1.1 Cell recovery:

　　According to customer needs, relevant cells are recovered. The recovery and culture of each cell are performed in accordance with 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.

　　1.2 Preparation of reagents:

　　1.2.1 Prepare 2x cell culture medium: Dissolve 2 g RPMI 1640 powder culture medium in deionized water, adjust the volume to 100 ml, pass this medium through a 0.22 μm filter to sterilize, add 20% FBS and 2% double antibody , stored at 4℃, valid for one month.

　　1.2.2 Prepare agarose gel: weigh 0.5g agarose and add 50ml deionized water to make a 1% agarose solution; weigh 0.3g agarose and add 50ml deionized water to make a 0.6% agarose solution; Seal the Erlenmeyer flask with a sealing film and a rubber band, heat it in the microwave for 30 seconds, take it out and shake it well, then heat it until it boils and becomes clear. Sterilize in a sterilizing pot at 121°C for 30 minutes and store in a refrigerator at 4°C. It is valid for one month. If necessary, heat it in a microwave until it becomes clear.

　　2. Operational procedures

　　2.1 Bottom agar plating:

　　2.1.1 Heat 1% and 0.6% agarose gel in a microwave oven until it is clear and transparent, then place it in a 37°C water bath to lower it to a constant temperature; preheat the 2×1640 complete culture medium water bath to 37°C.

　　2.1.2 Mix 1% agarose solution and 2× culture medium 1:1, 70 μl per well. Be careful not to press the pipette completely to avoid bubbles. Leave it on the operating table for 30 minutes until the agarose solidifies.

　　2.2 Upper agarose plating:

　　2.2.1 Digest and centrifuge the target cells, resuspend them for counting, and dilute them with 2× culture medium to the required concentration. Generally, 500 to 1,000 cells are spread in each well of a 96-well plate.

　　2.2.2 Mix the 0.6% agarose solution and the cell suspension at a ratio of 1:1, and add it to the solidified 96-well plate, 140 μl per well. Be careful not to hit the pipette to the bottom to avoid the generation of bubbles, and let it stand on the operating table. Wait 40 minutes until the mixed agarose solidifies.

　　2.2.3 Add 280μl PBS solution to the wells surrounding the cells.

　　2.3 Replenishing the upper culture medium:

　　After the agarose solidifies, add 50 μl of 1×1640 complete culture medium to each well, and replenish or change the medium every 3 days.

　　3. Medication and CTG detection

　　3.1 Observe whether the cells grow into monoclonal clones. This process takes about 1 week;

　　3.2 Add medication, process and culture according to customer requirements, and strictly implement it in accordance with relevant SOP;

　　3.3 Take out and aliquot the CTG from -20℃ and thaw it at room temperature, take the 96-well plate from the incubator to room temperature and equilibrate it for 1 hour;

　　3.4 Add 60 μl CTG, keep away from light on a shaker for 2 hours, mix with a volley gun, pipet out 100 μl into a 96-well white bottom plate, and read with a microplate reader;

　　3.5 Data analysis.