Considerations for Live Cell Analysis

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One of the many challenges of live cell analysis is ensuring that the actual assay environment is suitable for the proper cell growth and experimental result. Because “live cell analysis” uses living cells as opposed to fixed, dead, cells the challenges facing an experimenter can be daunting.

Assay Development Flow Chart
The challenges can be divided into two groups: Extrinsic, defined as those arising outside of the particular cell such as environmental temperature, and intrinsic challenges, defined as those that arise from within the cell or organism itself such as its rate of respiration.

Extrinsic Assay Elements:

Typical Live Cell Parameter Ranges:

Extrinsic Factor
Typical Ranges
Atmosphere Air or 5-7 Percent CO2
Humidity 97-100 Percent
Osmolarity 260-320 mosM
Oxygenation At least 20% O2 actual gas pressure in liquid will be lower.
pH 7.0-7.7
Temperature 35-37°C

Intrinsic Assay Elements:

  • Metabolic rate
  • Cell cycle
  • Potency
  • Size
  • Adherence
  • Migration
  • Cellular origin
  • Proliferation rate
  • Robustness
  • Density dependence growth

Optimal Intrinsic Factor Values:

Key Intrinsic Factor
Desired Quality



Size 15-20 microns
Adherence Yes, typically easier to maintain in small chambers
Growth rate/metabolic rate Moderate to slow, typically easier to maintain
Robustness Yes
Density dependent growth Not required, if at all possible
Growth Matrix/requirements Well Characterized

We create a more ideal experimental situation by monitoring or controlling key extrinsic factors including even the mechanical forces of the culture devices themselves. The resulting stability may then also help control some intrinsic characteristics of the cells or organisms themselves.

For example, cellular differentiation and developmental studies represent an important arena for live cell assays but such differentiation experiments are keenly sensitive to changes in the proliferation rates of the target cells, and such rates can vary with the age and passage number of the cell. Similarly, what stage of the cell cycle a particular cell or population is in influences not only when it will divide but its rate and gene expression too. These intrinsic factors may also be influenced by nutrient availability or other changes in environmental parameters. Thus, all of these factors need to be carefully considered and controlled as much as possible to ensure a successful experiment.