How to Choose the Correct Fluorochromes for Use with the NC-3000

Background

The NucleoCounter® NC-3000™ is an automated full-spectrum fluorescence microscope with integrated data collection, analysis and reporting. The automated image cytometry technology from ChemoMetec provide advanced, fast and precise analysis.

Cells are labeled with antibodies tagged with fluorochromes, dyes, tags which have light scattering properties. When light from the LEDs hit the labeled cells, the scattered light from cells or particles is used for identification or quantitative measurement of physical properties. Labels, dyes, and stains can be used for multi-parametric analysis to understand more properties about a cell. 

Immunophenotyping is the analysis of heterogeneous populations of cells using labeled antibodies and other fluorophore containing reagents such as dyes and stains. Light emitted from fluorophores are in a spectrum of wavelengths, so combining multiple fluorophores may cause overlap. To add specificity, optical filters and dichroic mirrors are used to filter and move light to the detector.

Optical filters are designed as band pass (BP), long pass (LP), or short pass (SP) filters. 

Choosing the correct fluorochrome or dye, therefore, becomes very important for a successful experiment.

How to Choose the Correct Fluorochromes

  1. The selection of fluorochromes or dyes is dependent on. a. The availability of the Excitation LEDs in the instrument. b. The availability of the Emission Filters in the instruments
  2. The following is a table for the full list of possible LED/Emission filter combinations available in the NC-3000 and some possible fluorochromes and dyes used in each channel.

Note: The denotation of emission filter as e.g. Em530/15 means a bandpass filter that allows light of wavelength 530nm ± 15nm (515 nm – 545nm) to pass. 3. To select a fluorochrome or dye by carefully selecting a combination of LED/filter and matching the fluorochrome to the selection.

4. Several online sites that help in proper fluorochrome selection such as Spectra Analyzer, Biolegend.com may be used as well.

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