Cell flat cloning formation experiment

1, cell suspension preparation: Take a single-stage cultured cell in several days, digested with 0. 25% trypsin and Blowing into a single cell, retained with complete medium, and produced cell suspension. 2, Cell Formula: Dilute the cell suspension to a gradient multiple, inoculation with a gradient density of 50, 100, 200, 500, 1000, 2000 cells, respectively, inoculated with 1 ml 37 C preheating culture In the liquid, it is gently rotated, and the cells are dispersed evenly. Place the 37 C 5% CO2 environment, stand in the culture. 3, Cell Observation: A single cell was observed under a microscope to tend to clone visible clones in the petri dish. Abandon. The supernatant, be careful with PBS twice. 1 ml of methanol was added, fixed for 15 minutes. Then remove the fixture, add the appropriate amount of Giemsa to use the stained liquid for 10 minutes, then slowly wash the dyeing liquid with water, air dry, and take pictures. 4, clon count: Direct count of digits directly, and then calculate the cloning formation rate. Results Flat Clones Cloning Rate u003d Cloning number of vaccination cells x100%