Transmission of monolayer cells
The medium was removed, trypsin was added for brief action, incubated, cells were dispersed in medium, counted, diluted and re-inoculated.
Operation method
Scheme 13.2 Transmission of monolayer cells
Principle
The medium was removed, trypsin was added for brief action, incubated, cells were dispersed in medium, counted, diluted and re-inoculated.
Materials and Instruments
A549 cells WI-38 MRC-5 Trypsin D-PBS Move 1. Prepare the ultra-clean bench by placing the reagents and materials on the ultra-clean bench to start the experiment. Caveat In each case, the primary dissociation reagent, whether trypsin or EDTA, is left in place only briefly to remove most of the digestate, leaving only a small amount for incubation. If difficulties are encountered in the isolation of cells and the subsequent preparation of single-cell suspensions, other procedures may be used. Common Problems Step 15 of this procedure should not be used as a long-term solution to the problem of elevated pH after passaging, but if the problem persists, reduce the pH of the medium as it is being prepared and check the pH of the medium in the incubator or in aerated flasks. For more product details, please visit Aladdin Scientific website.
70% Ethanol Sprayer
Growth media Suction tubes Centrifuge tubes Culture flasks Pipettes Suction earballs Suction paper towels Suction buckets Anti-ethanol markers Hematocrit plates
2. Carefully examine the culture for any signs of contamination or deterioration.
3. Based on the criteria and your knowledge of the culture's behavior, decide whether or not you need to pass it on (do Exercise 13 to record cell density and status, e.g., mitotic, multilayered, denatured cells). If passaging is required, proceed as follows.
4. place the culture in a sterile work area and discard the old medium. Separate the various cells (A 549 cells, inoculated at 2 x 104 /ml in 25 cm2 flasks for 7 and 14 days; WI-38, MRC-5, or the same normal diploid fibroblasts, inoculated at 2 x 104 /ml in 25 cm2 flasks for 7 and 14 days), and repeat the following steps for each cell.
5. To avoid washing out the cells, D-PBS/EDTA ( 0.2 ml/cm2 ) was added along the cell-facing side of the culture flasks (D-PBS containing 1 mmol/L EDTA), and the cells were washed to remove the pre-wash solution. The purpose of this step is to remove residual serum, which inhibits the action of trypsin and removes divalent cations required for cell adhesion.
6. Add trypsin (0.1 ml/cm2 ) (0.25 % , D-PBS configuration) along the cell-to-cell side of the culture flask. Turn the culture flask over and lay it flat. Ensure that the trypsin completely covers the cell layer. Allow to stand for 15-30 s.
7. Stand the flask up so that the trypsin leaves the cell layer and quickly check if the monolayer of cells has not been detached. If cell detachment is a problem before the cells have dissociated, the use of 4°C trypsin will prevent this problem.
8. Aspirate away most of the trypsin, leaving only a few drops.
9. Incubate the flask flat until the cells become round and bulge, tilt the flask and the monolayer of cells will slide off the surface of the flask (usually after 5-15 min). Be careful not to digest for too long, on the other hand do not force the cells to blow down before they are well digested, this will result in the cells falling off in sheets.
10. Add culture solution (0.1~0.2 ml/cm2 ) , repeatedly with a pipette (pipette with graduated, plugged cotton. If glass, place in a square cell box according to size, if plastic, individually packaged and sorted on a shelf for sucking off culture solution un-cotton plugged straws, if a pump and vacuum suction line are available) blowing on the cell surface of the monolayer culture to disperse the cells.
11. Finally, place the tip of the pipette in the bottom corner of the culture flask and blow the cells up and down several times, taking care not to create air bubbles. The intensity of blowing varies from cell line to cell line. Some cell lines are easily dispersed, while others need to be blown vigorously to be dispersed. However, if the blowing force is too strong almost all cells will be mechanically damaged by shear. Primary or early generations of cultured cells are particularly susceptible to damage due to their fragility and large size, while continuous cell lines are usually more resilient and need to be blown vigorously to fully dissociate. Blow well up and down to disperse the cells into a single-cell suspension. If this step is difficult, use a stronger dissociation reagent. 
Single-cell suspensions are preferred during passaging to help ensure accurate cell counts and uniform growth after re-inoculation. Preparation of single-cell suspensions is necessary for counting cell proliferation or labeling rates or for clonal isolation of cells.
12. Cells are counted and values recorded using a blood counting plate or electronic cell counter.
13. Dilute the suspension to the appropriate inoculum concentration.
(a) Add the appropriate amount of cell suspension to a culture flask that already has a known volume of medium (growth medium, e.g., 1 x MEM plus Earle's salt, 23 mmol/L NaHCO3, no antibiotics), or
(b) dilute the cells to the desired total volume and then dispense into individual culture flasks.
Procedure (a) is suitable for routine passages where the number of flasks is small and accurate cell counting and proliferative capacity assays are not required, but if multiple identical cultures are done at the same time, procedure (b) is more suitable. Because the number of operations is reduced, the cell density in each culture flask is exactly the same.
For Exercise 13, use procedure (b): dilute the cells in each flask with 20 ml of culture medium to 2 × 104/ml, take 5 ml of cell suspension from each flask, and inoculate it into 2 culture flasks.
14. If the cells are grown at elevated CO2 levels (e.g. Eagle MEM with Earle's salt and 23 mmol/L NaHCO3 as listed in the material), inject the correct gas (in this case 5 % CO2 ) from a pre-mixed gas mixture bottle or gas mixing device through a filter tube to the top of the medium in the culture flasks. Be careful not to blow the gas into the medium, as this will create air bubbles, which may spoil some components of the medium and also increase the chance of contamination. This step can be omitted if the normal gas phase is air, using Eagle MEM medium with Hank's salt.
15. Cap the flask, return it to the incubator, and check the pH change after approximately one hour. If the pH of the medium rises in the air phase, move the flask to a sterile area and rapidly (1-2 s) blow 5 % CO2 into it . Since the behavior of each culture is known in the same medium, it will eventually become clear which cells need to be blown during passaging, without having to be incubated first for observation. If the pH of the medium is still elevated in the 5 % CO2 gas phase (as in this experiment), increase the CO2 concentration to 7 or 10 % or add sterile 0.1 mol/L HCl.
16. Repeat the procedure from step 4 for the second cell line.