Cell Counter

At the Cell Counter: A431 Cells

The epidermoid carcinoma cell line A431 expresses abnormally high levels of epidermal growth factor receptor (EGFR) and is a useful model for studying EGFR-mediated signaling, which regulates growth, survival, proliferation, and differentiation in mammalian cells. Derived from the epidermis of an 85-year old female patient and established by D. J. Giard et al., A431 cells have proven to be a valuable model system for examining the cell cycle, apoptosis, and cancer.

View StainFree Cell Detection Webinar

Download StainFree Cell Detection Application Note

Download eBook: Count Cells Like a Pro

Figure 1 : Images obtained using SpectraMax MiniMax cytometer

A431 cells were stained with EarlyTox™ Live Red Dye and then imaged using the SpectraMax MiniMax cytometer. Cells were identified with a user-defined custom setting. Top: overlaid transmitted light and red fluorescent images; Bottom: cells identified using StainFree analysis (purple masks show cells identified by the software).

Figure 2: StainFree cell counts vs. fluorescent cell counts

A431 cells were seeded at densities ranging from 156 to 20,000 cells per well, and nuclei were stained using EarlyTox Live Red Dye. Images were acquired from 4 sites per well, and a region of interest in the middle of the wells was selected for analysis. Cells were then counted by using either the StainFree method (blue plot) or by counting red-fluorescent nuclei (red plot).

Figure 3: EarlyTox Caspase-3/7 NucView 488 Assay

A431 cells were treated with serial dilutions of staurosporine (red plot), camptothecin (green plot), and etoposide (blue plot) for 24 hours and then assayed for apoptosis using the EarlyTox™ Caspase-3/7 NucView 488 Assay Kit. Cells expressing caspase-3/7 were stained green fluorescent and were imaged and counted using the SpectraMax MiniMax cytometer. Results were plotted as apoptotic cell count vs. compound concentration using SoftMax Pro Software.


A431 cells have an epithelial morphology and tend to grow in clusters, making them a bit challenging to count. For StainFree counting, use ‘Create New Setting’ and use the yellow drawing tool to draw around the outlines of the cells without going outside the cell edges. Use the blue drawing tool to indicate non-cell areas and any artifacts.

A431 Cells Analysis Toolkit

Setting for cell counts
Optical configuration
SpectraMax MiniMax 300 Imaging Cytometer
Read mode
Read type
Wavelength settings

Transmitted light (TL)

713 nm (red fluorescence)

Image acquisition settings

TL exposure: 7 ms

TL Focus adjustment: 0 µm

713 exposure: 12 ms

713 Focus adjustment: 20 µm

Image analysis settings

Analysis type: Discrete Object Analysis

Wavelength for finding objects: TL (StainFree) or 713 (red nuclei)

Find objects

TL: Create new setting (Custom)

713: Set Size and Intensity: Size = 10-30 µm, Intensity Above Background = 70

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/i3x 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.