Inoculation of suspension cells in cell culture flasks

Summary

Inoculation of suspension cells in cell culture flasks can be used for (1) mass culture of cells and (2) providing cells with a suitable environment for growth. This experiment is from Cell Lab Guide (Previous) by Peitang Huang.

Operation method

Inoculation of suspension cells in cell culture flasks

Principle

Lymphoid cells usually grow in suspension. A commonly used culture medium is RPMI1640 with 10%~15% FBS. The optimal inoculum density and plateau cell density varies among cell lines. When culturing a new cell line the optimal principle is to passage the cells at 1:2 and 1:4 ratios and count the cells within 3-4 days to calculate the number of cells per milliliter. The number of cells will double 24-36 hours after passaging. If the cell density of the passaged cells is too low, the cells are prone to die, reflecting the long retention period of the cells before they reach growth. Once the cells are acclimatized to the culture conditions in the laboratory Ran decides on the optimal inoculum density for the prepared cell line. The number of cells inoculated per milliliter can vary from 5x104 to 8x105 The pH of the culture medium drops and turns yellow, indicating that the cells have reached maximum density and need to be replaced or passaged. Suspension of the cells is usually done by simple dilution, and if a complete change of culture medium is required, the cells can be precipitated by low-speed centrifugation and resuspended in a suitable culture medium.

Materials and Instruments

Lymphoid cells
RPMI1640 culture medium with 10%~15% FBS
Cell Culture Flask

Move

1, If the cells are not cultured in a rotary flask, simply shake, scrape, or trypsinize (see “Trypsin treatment to isolate cells” below) to separate the cells from the surface of the cell vials.

2, Blow the cells gently (up and down of the pipette) to break up the clumps of cells.

3, Take 1 ml of the cell suspension for cell counting and do not allow the cells to settle Do not allow the cells to be deposited at the bottom of the bottle. Gently rotate the vial back and forth to keep the cells in suspension.

4. Dilute the cells to a final concentration of approximately ^2x105~4x105 per ml.

5. Perform cell counting and cell viability testing with the Taipan blue rejection assay, as described in the protocol below & ldquo;Cell Counting”.

Caveat

Pay attention to asepsis and wipe hands with alcohol before handling.

Common Problems

Advantages of suspension cell culture: fast multiplication, can produce groups of cells of uniform age.

Source "A Guide to Cell Experiments (Previous Book)" by Huang Peitang.

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