HepG2 is a cell line derived from the liver tissue of a patient with hepatocellular carcinoma (HCC). It is often used as a model system for HCC as well as for studies of drug metabolism and toxicity. Cultures are adherent, with epithelial morphology, and tend to grow in small aggregates that make the counting of individual cells difficult. Under the right culture conditions, they can form distinct apical and basal surfaces amenable to studies of liver disease.
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Figure 1: StainFree determination of % covered area
HepG2 cells were imaged using the SpectraMax MiniMax 300 Imaging Cytometer and cells were identified using a custom, user-defined analysis setting. Shown on the left are the original transmitted light images, and on the right is the same image with purple masks indicating cells identified by the software. Two cell densities are shown for comparison.
Figure 2: StainFree vs. fluorescent analysis of % covered area
Confluence of HepG2 cells seeded at densities ranging from 234 to 30,000 cells per well was analyzed using StainFree technology (blue dots), or they were stained with EarlyTox™ Live Cell Assay dye and fluorescent area was analyzed (green dots). The percent area covered for a region of interest of the image for both methods agreed closely across the entire range of cell densities.
Figure 3: EarlyTox Caspase-3/7 NucView 488 Assay
HepG2 treated with media control (left panels) or 200 µM capsaisin (right panels) were assayed with the EarlyTox Caspase-3/7 NucView 488 Assay Kit and imaged using the SpectraMax MiniMax 300 Imaging Cytometer with green fluorescent and transmitted light (TL) channels. Top row: fluorescence image showing apoptotic cells labeled with green fluorescence. Bottom row: overlaid TL and fluorescence images showing minimal apoptosis in control cells and nearly 100% apoptosis in treated cells.
Figure 4: EarlyTox Caspase-3/7 NucView 488 Assay (time course)
HepG2 treated with 200 µM capsaicin (left bar), or media control (right bar), for 4.5 hours (red bars), 6 hours (green bars), or 22 hours (blue bars). Apoptosis was assessed using the EarlyTox Caspase-3/7 NucView 488 Assay Kit. Cells were incubated with 5 µM of NucView 488 substrate and imaged at the time points indicated.
Figure 5: EarlyTox Caspase-3/7 R110 Assay Kit and EarlyTox Glutathione Assay Kit
HepG2 treated with 200 µM capsaicin (left bar of each pair) or media control (right bar of each pair) were assayed using the EarlyTox Caspase-3/7 R110 Assay Kit (red and green bars), which measures caspase activity, or EarlyTox Glutathione Assay Kit (blue bars), which measures the decrease in glutathione, an early indicator or apoptosis. Both assays were detected on the SpectraMax i3x Multi-Mode Microplate Reader using preconfigured protocols in SoftMax Pro Software.
Tip: HepG2 cells grow in aggregates or clumps of irregularly shaped cells that are indistinguishable by eye. A useful way to quantify HepG2 cell growth or confluence is by using StainFree™ Technology to determine the area covered by cells. Images collected in the TL channel can be easily analyzed using the Field Analysis setting. The predefined ‘Cells’ analysis setting can be used, or a custom analysis can be designed by using the drawing tools in SoftMax Pro Software.
HepG2 Cells Analysis Toolkit
- SpectraMax® i3 Multi-Mode Microplate Detection Platform
- SpectraMax® MiniMax™ 300 Imaging Cytometer
- SoftMax® Pro Software
Instrument Settings
| Parameter | Setting for cell counts |
| Optical configuration | SpectraMax MiniMax 300 Imaging Cytometer |
| Read mode | Imaging |
| Read type | Endpoint |
| Wavelength settings | Transmitted light |
| Image acquisition settings | Exposure: 7 ms (TL), 5 ms (541) |
| Image analysis settings | Analysis type: Field Analysis Wavelength for finding objects: TL |
| Find objects | Settings: Custom setting |
About StainFree Cell Detection Technology
Imaging cell-based assays typically requires the use of fluorescent probes that can be toxic to living cells or may only function in fixed cells. A label-free method for analyzing cell counts and cell confluence enables researchers to quantitatively monitor cell proliferation and health without time-consuming workflows that may disrupt cell viability.
The SpectraMax i3 Multi-Mode Microplate Platform with MiniMax 300 Imaging Cytometer uses unique, patent-pending StainFree Cell Detection Technology that allows you to perform cell proliferation, cytotoxicity, and other assays without nuclear stains like DAPI, which intercalates with DNA, or live cell dyes that are actually toxic to cells in the long term.