Chapter 2: Experimental Design
Sample Preparation
Experimental Design
The FlowSight
®
System can quantify the intensity, specific location, and distribution of signals within tens of thousands of
cells per sample. The system can perform most standard flow cytometric assays, and can also leverage the technology's
imaging capabilities to discriminate image-based changes within individual cells and cell populations.
l
Choice of Cell Type: The cell/particle size should be less than 60 microns in diameter.
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Final Sample Concentration and Volume: At least 1 million cells in 50 μl (2x10
7
cells/mL) in PBS/2%FBS in a 1.5
mL siliconized microcentrifuge tube.
l
Protocols: In general, any established labeling protocol used for flow cytometry will work with the FlowSight
System (see Current Protocols in Cytometry for general labeling techniques). Stain cells on ice in the presence
of azide when possible to reduce non-specific capping of antibody. Use polypropylene tubes, preferably sil-
iconized, to process samples.
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Choice of Fluorochromes: Choose fluorochromes that are excited by the lasers in your FlowSight System (405,
488 and 642 nm are most common). Use the chart below or look online for a spectra viewer that will help you
plan which dyes will work best.
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Compensation: Have a sample of cells each labeled with a single-color for each fluorochrome used (i.e., FITC
only cells, PE only cells, etc.)
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Cell Aggregation: Minimize aggregation problems by straining the sample through a 70 micron nylon mesh
strainer or by using an anti-clumping buffer such as EDTA or Accumax
™
prior to fixation.
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Fixation: If fixation is desired, thoroughly fix cells with 1% formalin on ice for 20 minutes.
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Number of samples: No more than 30 total for feasibility experiments. Please limit the samples to the fol-
lowing; Positive and Negative biologic controls, compensation controls, and experiment samples.
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Brightness of Stain and Stain Balancing: Quantifying the location and distribution of signals in an image is a
demanding task that requires optimized labeling.
Here are a few suggestions to help design the experiment:
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Try to achieve at least a full log shift in fluorescence, as measured by a standard flow cytometer.
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Use the brightest dye for the antigen with the smallest copy number
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The brightness of probes can be independently controlled by changing the laser power.
However, data quality is enhanced when the brightness levels of all probes excited off a single
laser are balanced to within a log of each other. Probe balancing avoids the saturation of bright
stains when they are combined with dim stains in the same sample.
For Research Use Only. Not for use in diagnostic procedures.
6
Amnis
®
FlowSight
®
Imaging Flow Cytometer User Manual
