5. Count Apps
Count apps are designed for a common cell counting techniques such as Brightfield, Trypan Blue,
AO/PI, etc. To properly count a sample and optimize a result, the researcher must first capture the
best image(s) possible. Examples of proper and improper adjustments are included in this guide in
Section 6, Count Tab.
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Protocols and Optimizing Settings
Each Count app includes the relevant group of count settings saved as a Protocol. Once a sample has
been counted, protocol parameters can be adjusted using the Optimize Settings button..
Channels
Count apps use an application specific set of optical channels. The CellDrop FL model includes
Brightfield, Green Fluorescence and Red Fluorescence channels. The CellDrop BF model includes
Brightfield only.
The Brightfield app is used for quick, label-free total cell or particle counts. A brightfield count does
not determine cell viability. This app will count cells with a bright, white center and a defined border.
The application is not designed to count dark, solid objects.
Trypan blue is a cell membrane impermeable dye that only enters cells with compromised
membranes. Upon entry, the dye binds to intracellular proteins resulting in bluish colored cells. The
trypan blue exclusion assay enables a direct counting of live (unstained) and dead (stained) cells.
Published literature suggests that using trypan blue for samples with viabilities less than 70% may
result in higher reported cell viability values when compared to fluorescent-based detection methods.
% Viability =(Live Cell Count / (Live Cell Count + Dead Cell Count)) *100.
The AO app uses green fluorescence images to count live nucleated cells.
Acridine orange is a nuclear staining cationic fluorescent dye that enters live and dead cells and
interacts with DNA and RNA by intercalation or electrostatic attractions. When bound to DNA, it is
spectrally very similar to fluorescein, with an excitation maximum at 502 nm and an emission
maximum at 525 nm.
Although AO enters dead cells, the intensity of the green fluorescence is very low for these cells
compared to the green fluoresce emitted from live cells. This app may be useful when counting
solutions containing either red blood cells and/or significant cellular debris.
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