Growth curve with a monolayer culture in multiwell plates

Outline :
Set up a series of multiwell plates with cultures at three different cell conc and count cells in one plate at daily intervals until the culture reaches plateau phase.

Materials :

monolayer cell culture : like A54 9, Vero , Hela-S3 , 75 cm2 with late log phase
Trypsin 0.25 % crude wit h 10mM EDTA 5ml
Growth medium with 4mM NaHCO3
D-PBSA
Flask 25 cm2

Protocol:
Trypsinize cells as for regular subculture
Dilute cell suspension to 1X105 cells/ml, 3X104 cells/ml, and 1X 104 cells/ml in 25 ml of medium for each conc.
Seed three 12-well plates with 2 ml of 1 X 104 cells/ml suspension to each well of top 4 wells, 2 ml of 3X104 cells/ml to each well of the second row and 2 ml of 1X105 cells/ml to each well of third row. Add cell suspension slowly from center of well so that it does not swirl around well. Similarly do not shake plate to mix the cells, as circular movement of medium will concentrate cells in the middle of well.
Place plates in a humid CO2 incubator or sealed box gassed with 5% CO2.
After 24hr remove first plate from incubator and count cells in three wells at each concentration,
Remove medium completely from three wells containing cells to be counted
Add 0.5ml of trypsin /EDTA to each of three wells
Incubate plate for 15min
Add 0.5ml medium with serum, disperse cells in tryspin /EDTA/medium and transfer 0.4ml of suspension to 19.6 ml of D-PBSA.
Count cells on electronic cell counter

Stain cells in remaining wells at each cell density
Repeat sampling at 48 and 72 hr as in step 5 and 6
Change medium at 72 hr or sooner if indicated by a drop in pH.
Continue sampling daily for rapidly growing cells (i.e.e cells with PDT of 12-24 hr) but reduce frequency of sampling to every 2 days for slowly growing cells (i.e. cells with PDT > 24 hrs) until plateau phase reached.
Keep changing medium every 1,2 or 3 days, as indicated by fall in pH.

Analysis of monolayer growth curves:

Primary count: cells/ml
Cell/flask OR well
Cell concentration: cells/ml of culture medium
Cell density: cells/cm2 of growth surface
Plot cell density (cell/cm2) and cell conc (cells/ml) both on log scale against time on a linear scale.
Determine lag time, PDT and plateau density
Establish appropriate-starting density for routine passage. Repeat growth curve at diff cell conc if necessary
Complete growth curves under different conditions and try to interpret data
Examine stained cells at each density to

1. Determine whether the distribution of cells in flasks/wells is uniform and whether cells are growing up the sides of wells
2. Observe differences in cell morphology as density increases
3. Compare cell-cell interaction in normal and transformed cells