Cell Counter

At the Cell Counter: CHO Cells

Chinese Hamster Ovary (CHO) cells are an epithelial-like cell line commonly used in biological and medical research. Developed over 50 years ago, they have been used in innumerable studies ranging from genetics to cytotoxicity. They are highly amenable to transfection and have become the most popular cell line for manufacturing recombinant proteins, including therapeutics.

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Figure 1. StainFree analysis of CHO cells using SoftMax Pro Software

CHO cells in these images were plated at 4000 cells per well in a 384-well microplate. Left: To create a new StainFree analysis setting, the mouse is used to ‘draw’ on the image, indicating individual cells (yellow) or non-cellular areas (blue). Right: Purple masks show the objects identified in the image by the software. Alternatively, a predefined image analysis setting can be used for one-click analysis of CHO cell counts.

Figure 2. StainFree technology compared to fluorescencecell counting

Cells counted using StainFree™ Cell Detection Technology (blue), red nuclear stain (red), and green whole-cell stain (green). Counts obtained from all three methods agree closely, demonstrating that StainFree technology gives accurate cell counts while eliminating the need for fluorescent dyes (r > 0.99 for each plot).

Figure 3. Cell confluence

Percent confluence of cells just prior to transfection was determined using StainFree technology. For initial seeding densities of 15000, 10000, and 5000 cells per well, the measured percent confluence after overnight growth was 44, 30, and 15, respectively.

Figure 4. Imaging of GFP-transfected cells

Transfected cells were imaged using the TL (A, C) and green fluorescent (B, D) channels of the SpectraMax MiniMax 300 Imaging Cytometer. Cells shown were seeded at 15000 cells per well in a 96-well plate. Two transfection conditions are shown, representing low (B) and high (D) transfection efficiencies. StainFree technology was used to identify individual cells, which could then be classified as positive or negative for GFP expression.

Tip 1:

To count CHO cells without staining, I use the predefined setting ‘Cells A’ in SoftMax Pro Software. This works really well on CHO cells that are anywhere from 5% to 95% confluent. We’ve been able to get excellent correlation between Stain-Free counts and fluorescent nuclear counts from the very same samples. (See Fig. 2 & 3).

Tip 2:

Getting more accurate StainFree counts. When imaging in the TL channel, instead of using a Focus Adjustment height that gives you the sharpest image possible, try lowering your Focus Adjustment to a somewhat more negative position, e.g. from 0 to -20. You want the cells in your image to have bright centers and dark edges. This will save you lots of time and give you better results with the predefined analysis settings.

CHO Cells Analysis Toolkit

Instrument Settings

Optical configuration
SpectraMax MiniMax 300 Imaging Cytometer
Read mode
Read type
Wavelength settings
Transmitted light
Image acquisition settings
Exposure: 7 ms
Image analysis settings
Analysis type: Discrete Object Analysis Wavelength for finding objects: TL
Find objects
Setting: CellsA
The ‘CellsA’ predefined image analysis setting enables a simple one-click analysis of CHO cell counts. Image acquisition and analysis settings may vary depending on assay conditions, microplate used, and other factors.

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.