Experiments on the preservation and culture of insect cells
Experiments on the preservation and culture of insect cells
Grasshopper nightshade cells can be cultured either in monolayer or suspension (rotary) cultures, and can be preserved and passaged in serum-containing cultures, or in serum-free cultures.
Operation method
basic program
Materials and Instruments
Insects Move 1. Add 4 ml of complete insect cell culture medium containing 10% fetal bovine serum to a 25 cm2 culture flask. Remove an ampoule of frozen Sf9 cells, quickly place it in a 37°C water bath, and shake it vigorously back and forth by hand to melt it. When the contents of the ampoule are almost completely melted, dip the ampoule into 70% ethanol to sterilize the outer wall. For more product details, please visit Aladdin Scientific website.
Fetal Bovine Serum, Typhon Blue, Ethanol
Culture Bottle Incubator Cell Counter Stirring Table Rotor Centrifuge
2. Break the neck of the ampoule, transfer the content to a 25 cm2 culture flask, shake the flask gently by hand to disperse the cells evenly, and incubate the cells at 27℃ for 2~3 h until the cells adhered to the wall.
3. Remove the old culture medium and replace with 5 ml of fresh complete culture medium containing 10% fetal bovine serum. Continue to incubate and change the medium every 3 days until the cells grow in confluent pieces.
4. Prepare a new 25 cm2 culture flask with 4 ml of complete culture medium containing 10% fetal bovine serum.
5. When Sf9 cells have grown in confluent sheets, discard the culture medium from the culture flask. Add fresh complete culture medium and resuspend the cells by gently blowing with a pipette or tapping the culture flask with the palm of your hand.
6. Cells were counted using a blood cell counter designed for culturing tissue cells by inoculating 25 cm2 culture flasks with 1×106~2×106 cells and shaking the flasks to distribute the cells evenly. The culture flasks were incubated at 27°C and changed with complete medium containing 10% FBS every 3 days until the cells in the flasks grew to confluence.7. Discard the culture medium from the monolayer of cultured cells in the confluent piece and resuspend the cells.8. Count the cells using a blood cell counter. Inoculate the cells in a rotary culture flask at a density of about 4×105~5×105 cells/ml. Incubate at 27°C with constant speed agitation at 60~80 r/min. Slightly open the side vent to ensure sufficient air.9. Count the cells every 2-3 days and pass on when the cell density reaches 2×106~2×106 cells/ml, transfer the appropriate amount of cells to a new culture flask containing fresh complete culture medium to reach a final density of 4×105~5×105 cells/ml. alternatively, pour out the cell suspension from the appropriate body hedgehogs and replace it with fresh culture medium.10. Determine cell viability by adding 0.1 ml of 0.4% Tapan Blue to 1 ml of logarithmically grown cells. Examine the cells under a low-power microscope, count the cells stained with TPB (dead cells), and count the total number of cells.11. Pass the cells into a mixture of 1 part complete culture medium/10% fetal bovine blood supernatant and 1 part serum-free culture medium. Allow the cells to grow to confluence (monolayer culture), or to reach a density of 2×106~3×106 cells/ml (suspension culture).12. The cells were passaged into a mixture of complete culture medium containing fetal bovine serum in a ratio of 1:4 to serum-free culture medium and allowed to grow.13. Repeat the steps of cell passaging and cell growth with a mixture of complete culture medium and serum-free culture medium at a ratio of 1:8 to 1:10.14. Passage of cells in serum-free culture medium.15. Count the exponentially growing cells to be frozen, centrifuge at 1,000 g for 10 min at room temperature and discard the supernatant.16. Resuspend the cell pellet with complete culture medium containing 10% fetal bovine serum at a density of 1×107~2×107 cells/ml. Add equal volumes of complete culture medium containing 10% fetal bovine serum and 20% DMSO and place the cells in an ice bath. 1 ml of cells were pipetted into a freezing tube with a screw cap and stored at -20°C for 2 h, then at -70°C overnight.17. Transfer frozen cells to a liquid nitrogen tank for long-term storage.