Cell Proliferation and Viability Assays

Cell proliferation is a process where cells grow and divide in order to replenish cells that have died or to expand a population. While this process is tightly controlled in heathy tissue, a hallmark of cancer is dysregulation of the cell cycle, leading to an imbalance of cell growth. The ability to accurately measure cell proliferation assays are a key tool in cancer research and drug discovery studies. The BioTek Lionheart FX Automated Live Cell Imager and Cytation Cell Imager Multi-mode Readers support diverse methods for conducting cell proliferation and viability assays by directly determining the number of cells within a population.


High Contrast Cell Counting—Label-free direct cell counting

Label-free direct cell counting techniques are a convenient and effective alternative to methods that rely on fluorescent labels or indirect biochemical readouts of cell population size. The High Contrast (HC) cell counting application, available with BioTek Lionheart FX Automated Live Cell Imager and Cytation Cell Imager Multi-mode Readers, uses modified high contrast brightfield to conduct accurate label-free cell counts.

High Contrast Brightfield
High Contrast Brightfield

Cell Proliferation
HC Cell Counting

Cell Proliferation
Overlaid Images with Object Masks

Cell Proliferation
Strong Linear Correlation with Counts Derived from Nuclear Label-Based Method

Figure 1. High Contrast (HC) Cell Counting uses modified high contrast brightfield imaging to generate a distinct bright signal corresponding to each cell that is readily identified and counted using Gen5 image analysis tools. HC Cell Counts are comparable to counts derived using fluorescent nuclear labels across a range of cell densities (HT1080 data shown)


Key advantages of High Contrast Cell Counting

  • Convenient.  Accuracy and sensitivity of direct cell counting without the limitations associated with fluorescent labels
  • Robust.  Automated image analysis produces results comparable to nuclear stains across wide range of cell densities
  • Insightful.  Enables detailed phenotypic characterization of cell morphology and viability  


Determination of population size using both direct cell counts and % confluence

High contrast brightfield images and Gen5 image analysis tools are used to quantify the size of a cell population

Figure 2. High contrast brightfield images and Gen5 image analysis tools are used to quantify the size of a cell population. HT-1080 cells seeded within a 96-well microplate were imaged using the high contrast brightfield mode and 4x objective. The relative size of each population of cells is determined and reported as both % confluence and cell count within the imaged area.


Kinetic Proliferation and Viability Assay

A fully automated high throughput assay combining label-free HC Cell Counting and fluorescence imaging of markers for apoptosis and necrosis to monitor changes in both total cell number and cell death over time. The BioTek Lionheart FX Automated Live Cell Imager and BioSpa Live Cell Analysis System support multi-day proliferation and viability applications.

Automated kinetic analysis of proliferation.

Figure 3. Automated kinetic analysis of proliferation. (A) 384-well plate overview of cell proliferation. Red curves indicate cell count plotted over the duration of the 3 day experiment. As drug concentration increases from rows C through P, reduced proliferation is observed for all drugs across the entire 384-well plate. (B) Average cell count plots were overlaid for several concentrations of nocodazole (orange circles, n=12 replicate wells) and camptothecin (green circles, n=12 replicate wells) over three days, with Gen5™ generated dose-response curves for each drug treatment.


Quantitative analysis of apoptosis and necrosis normalized to total cell count

Combining label-free cell counting and fluorescent imaging of apoptosis and necrosis markers provides an accurate method for measuring treatment-induced cell death over time normalized to total cell count.

Figure 4. Fluorescence-based kinetic cell death analysis. (A) Example images of cells positive for pSIVA apoptosis marker and PI necrosis marker at two time. Positive apoptotic and necrotic cells are identified using size and signal threshold parameters with primary masks applied (inset shows enlarged region for clarity). (B) Kinetic profiles of % apoptotic and % necrotic are used to generate dose-response curves and EC50 values.


Increase assay throughput and performance

BioSpa™ Compatible Assay: Live-cell analysis on up to 8 microplates
The BioSpa Live Cell Analysis System
enables multi-plate kinetic analysis of cell proliferation and cell death


Application Notes:


Technical Note:

For Research Use Only. Not for use in diagnostic procedures.