Single-cell spreading plate is to add appropriate amount of complete medium into each well of cell culture plate in advance, shake well to make the medium infiltrate the whole bottom of the well, and then drop the weighted cell suspension, shake well to mix well and disperse the cells without clumping or excessive vacuolation.
Operation method
platelet
Principle
Single-cell spreading plate is to add appropriate amount of complete medium into each well of cell culture plate in advance, shake well to make the medium infiltrate the whole bottom of the well, and then drop the weighted cell suspension, shake well to mix well and disperse the cells without clumping or excessive vacuolation.
Materials and Instruments
DMEM medium, sterile PBS, 0.25% trypsin Move (1) Cultivate the cells for a period of time, and select the cells with good growth status and in logarithmic growth phase; (2) Discard the old medium and gently rinse with PBS for 3 times; (3) Discard PBS and add 1 mL of 0.25% trypsin to cover the cells; (4) Incubate the cells in a cell culture incubator for about 2 min to fully digest the cells; (5) Take out the cells and put them under the microscope for observation. Shake the petri dish to see the cells shrinking and rounding and floating slightly, then terminate the digestion in time by adding an equal amount of complete culture medium to terminate the digestion; (6) Mix gently, centrifuge at 1000 rpm for 5 min, and discard the supernatant; (7) Resuspend the cells with complete medium, count the cells and inoculate them into 6-well cell culture plates according to 6-8×105 cells/well. 1 mL of complete medium was added to each well in advance, gently mixed, and placed in the cell culture incubator for culture. Caveat (1) 30 min before the experiment, the cell room and the ultra-clean table should be sufficiently irradiated with ultraviolet radiation;(2) Enter the cell room to change the sterile gown, sterile slippers and so on； （3） Cell inoculation density 6-8×105(3) Cell inoculation density 6-8×10 5 cells/well, depending on the size of the plate, 60-70% of the wells should be spread； （4） Trypsin digestion time depends on the cell type, the initial experiment needs to set up a time gradient to find out； （5） Add the medium should be mixed well to ensure that the cells are evenly distributed, and will not be more or less in one place to show the phenomenon of clustering. For more product details, please visit Aladdin Scientific website.
Pipette, sterile tip, sterile EP tube, 6-well cell culture plate, cell counting plate