The ability to accurately determine cell number is an important aspect of a broad range of applications. The applications require setting up and optimizing cell-based assays, normalizing data across samples, and conducting cell proliferation assays.
The Agilent BioTek Lionheart FX automated microscope and Agilent BioTek Cytation cell imaging multi-mode readers utilize label-free and fluorescence imaging modes. Combined with powerful Agilent BioTek Gen5 image analysis tools, the systems deliver efficient and effective automated cell counting.
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 Lionheart automated microscopes and Cytation cell imaging multimode readers, uses modified high contrast brightfield to conduct accurate label-free cell counts.
HC cell counting: Modified high contrast brightfield imaging is used 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 (HT-1080 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
Kinetic proliferation assay: direct cell count, label-free
HC cell counting and Gen5 image analysis tools provide label-free quantification of cell proliferation over time. HT-1080 cells seeded within a 96-well microplate were imaged using the high contrast brightfield mode and 4x objective. The number of cells within the image area for each sample are reported using the HC cell counting application.
Kinetic proliferation assay: direct cell counts - fluorescence
Fluorescence imaging and Gen5 image analysis tools enable reliable detection and quantification of fluorescently labeled cells. Fluorescent nuclear labels enable direct cell counts of cell types that tend to cluster or present challenging morphologies. HCT-116 expressing an H2B-GFP label were seeded within a 96-well microplate and followed over a multi-day time course to measure cell number expansion. Fluorescence images were captured using the 4x objective. Gen5 image analysis tools are used to identify each nuclei (fuchsia masks) with total cell number per image area reported.
Detailed subpopulation analysis
Gen5 Image analysis tools provide accurate and efficient determination of cell population statistics. Cell counts and characteristics, including cell size and fluorescence intensity, are used to determine statistically-derived thresholds for conducting subpopulation analysis. The number of cells within the population responding to treatment (indicated by green fluorescence) are determined for each sample (fuchsia masks) using thresholds defined by negative control values (mean RFU + 2SD).
Data normalization using automated cell counts
Cell counts provide a straightforward method for normalizing results across sample conditions. Using Gen5 fluorescence imaging and cellular analysis tools, sample counts of Hoechst 33342 stained cells are used to normalize Agilent Seahorse XF metabolic data across 3 different cell densities.
Agilent BioTek Cell Count & Viability App
The Agilent BioTek Cell Count & Viability App is an efficient tool for automating mammalian cell counts, enabling rapid counts and producing high quality data. The App is a key component of the Agilent BioTek Cell Count & Viability Starter Kit. The kit is designed to help you get started with automated cell counting with your phase contrast-equipped Cytation cell imaging multimode readeror Lionheart automated microscope.
Key advantages of Cell Count & Viability App
- Higher quality data
- Automated % live/dead calculationsAgilent
- Built-in dilution calculator facilitates downstream applications
Learn more about Agilent BioTek cell counting applications - search our application notes and technical documentation in resources.
For Research Use Only. Not for use in diagnostic procedures.