Experiments for drawing growth curves of cells cultured in monolayers in multiwell culture plates
Experiments for drawing growth curves of cells cultured in monolayers in multiwell culture plates
Three sets of cells were cultured in multiwell plates at three different cell concentrations, and cells in one plate were counted each day before reaching plateau.
Operation method
Scheme 21.8 Experiment for plotting growth curves of cells cultured in monolayers in porous culture plates.
Principle
Three sets of cells were cultured in multiwell plates at three different cell concentrations, and cells in one plate were counted each day before reaching plateau. Move makings For more product details, please visit Aladdin Scientific website.
non-sterile
Monolayer cell culture: A549, Vero or HeLa-S3, 75 cm2 flasks, 1 late log phase
0.25% crude trypsin mixed with 10 mmol/L EDTA 5 ml
Growth solution, 100 ml, with 26 mmol/L NaHCO3 300 ml
D-PBSA (pre-washed and used for cell counting) 50 ml
10 12-well culture plates
Non-sterile:
Plastic box for plates
CO2 incubator or 5% CO2 to fill plastic boxes
Procedure
1. For normal passaging, first digest the cells with trypsin (see protocol 13.2).
2. Dilute the cell suspension to 1X105 cells/ml, 3X104 cells/ml and 1X104 cells/m l, each containing 25 ml of medium.
3. Use three 12-well plates with one cell concentration per well and 2 ml of cell suspension per well. Add 1X104/ml to the top four wells, 3X104/ml to the second row, and 1X105/ml to the third row (Figure 21. 7). Add the cell suspension slowly from the center of the wells so that the liquid does not vortex in the wells. Similarly do not shake the culture plate with a view to mixing the cells as the circular motion of the medium will concentrate the cells in the center of the wells.
4. Place the plate in a humidified CO2 chamber or fill the plastic box with 5% C02 and close.
5. After 24 h, remove the first plate from the incubator and count the number of cells in 3 wells for each concentration:
(a) Completely aspirate the culture medium from the 3 wells containing the cells to be counted;
(b) Add 0.5 ml of trypsin/EDTA to the above 3 wells;
(c) Incubate the plate for 15 min;
(d) Add 0.5 ml serum-containing medium, separate the cells in trypsin/EDTA/medium, and take 0.4 ml cell suspension into 19.6 ml D-PBSA;
(e) Cells were counted using an electronic cell counter.
6. Stain the cells in the remaining wells of different densities (see section 16. 4.2).
Tip It is also possible to use a hemocytometer plate for cell counting, but this is more difficult for lower cell concentrations. If a hemocytometer plate is to be used, reduce the volume of trypsin to 0.1 ml, carefully dispense the cells with a micropipette so that no air bubbles are produced, and then transfer the cells to the hemocytometer plate.
7. Repeat sampling at 48 and 72 h as in steps 5 and 6.
8. Change the culture solution at 72 h or earlier, depending on the drop in pH (see Scheme 13.1, Figure 22b).
9. For fast-growing cells (i.e., cells with a PDT of 12-24 h), sampling can be done every day, but for slow-growing cells (i.e., cells with a PDT > 24 h), sampling should be done every two days until the cells have reached a plateau.
10. Change the medium every 1, 2 or 3 days depending on the pH change. 
