Applications Guide: How to choose fluorophore combinations for Flow Cytometry

Wed, 06/14/2017 - 11:22


Flow cytometry is an experimental method that was developed to label and examine a high volume of cells in an extremely rapid rate using antibodies conjugated to fluorophores.  The basic concept of flow cytometry is that a cell suspension is situated into a single stream, which then passes through a light source that uses detectors to generate data sets based off cellular properties.  More specifically, the fluorescent light emitted by fluorophores conjugated to antibodies is channeled through selected filters to sort based off preset parameters or targets used. Flow cytometry is particularly useful in cell viability and proliferation assays, as well as diagnosing disease (particularly blood cancers).  When creating a panel of fluorophores for your flow cytometry panel, it is important to follow a few basic guidelines.

First, it is important to understand your flow cytometer.  You should know the type of laser in your instrument, the number of lasers present and their excitation capabilities, and how to set your filters.  Filters are important given that setting a filter too wide may lead to an excess of background noise, leading to false results.   Next, the desired fluorophores are selected. A good rule of thumb is to choose robust dye colors, as these will determine how prominent the signal is over the unstained cell population. It is, however, important to optimize any conjugation procedures to produce maximized results.  Using the stain index (D/W), where “D” is the difference between positive and negative populations and "W" is equal to 2SD of negative populations, can help determine if ratios are correct.  In addition, filling or supplementing a panel with a fluorophore with a robust dye is typically the best choice.  In some cases however, it may make more sense to choose a more dim dye in order to avoid spillover and loss of resolution and sensitivity.

The following is an example of a combination of fluorophores for a 6-color panel; FITC/Alexa Fluor 488, PE, PerCP-Cy5.5, PE-Cy7, APC/Alexa Fluor 647, and APC-Cy7/APC-H7).  If you are using an 8-color panel, you can use the above fluorophores and simply supplement with AmCyan and Brilliant Violet 421.  It is important to be conscious of the potential false positives produced by using tandem dyes, due to their tendency to show degradation.  These fluorophores are subsequently conjugated to an antibody target of choice. 

Flow cytometry HIF1a antibody

HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Analysis using the Alexa Fluor (R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in H929 cells using HIF-1 alpha antibody.

Novus Biologicals has an extensive list of conjugated antibodies available to meet your Flow Cytometry needs.  To read a comprehensive overview of Flow Cytometry and how it works from start to finish, please visit our page on "The Flow Cytometry Illustrated Assay".  To build your own sample Flow Cytometry panel, please take time to walk through our Panel Builder Tool. Furthermore, our antibody labeling kits will let you label almost any primary antibody in less than 30 seconds' hands-on time. 

View conjugated antibodies

 

  1. Chattopadhyay PK, Gaylord B, Palmer A, Jiang N, Raven MA, Lewis G, Reuter MA, Nur-ur Rahman AK, Price DA, Betts MR, Roederer M. Brilliant violet fluorophores: a new class of ultrabright fluorescent compounds for immunofluorescence experiments. [PMID: 22489009]
  2. Maecker HT, Frey T, Nomura LE, Trotter J. Selecting fluorochrome conjugates for maximum sensitivity. [PMID: 15536642]
  3. Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. [PMID: 16299475]

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