Monitoring Cell Culture Growth Kinetics
Kinetic growth profiles provide valuable information when designing live cell assays. This information includes determining the appropriate number of cells to load per well based on doubling time and cell size, as well as desired experiment duration (Figure 1.). This process can also be used to guide the optimization of cell culturing conditions, such as determining ideal serum and supplement concentrations.
A broad range of fluorescent stains are available for directly counting cells that are not amenable to label-free counting methods. The addition of these reagents can produce unintended cell type-dependent cytotoxic effects. Agilent BioTek Gen5 image analysis tools provide the ability to determine the appropriate concentrations of reagents to achieve efficient labeling through the experiment. This is done without significantly altering cellular behavior in the process. In the Figure 1. the HT-1080 cells were stained with a range of Hoechst 33342 concentrations. The treatment was done to determine an effective staining protocol to accurately measure treatment-induced changes in HT1080 proliferation rates.
Figure 1. The minimal amount of stain necessary to effectively identify and count each cell was determined by imaging HT1080 stained with a range of Hoechst 33342 concentrations over a 48-hour time course and then (A) calculating the signal-to-noise ratios (S/N) over time. (B) It was determined that a 0.032 mM concentration had no statistically relevant effect on HT1080 proliferation and provided satisfactory S/N (> 10) for accurate cell counting over the course of the 2-day experiment.