Typing “flow cytometry” into the Google search box returns 11,300,000 results! A daunting amount of information to process for anyone approaching the technology for the first time. To help anyone wanting to learn more about flow cytometry navigate this huge body of information before digging into the finer details of planning and designing their first experiment, we gathered a few helpful resources.
Training and Learning
Experiment Planning and Design
The first step is understanding the biology of the cell subpopulation under scrutiny. Once the cellular markers defining the subpopulations are identified, FluoroFinder’s panel builder can help match markers with fluorophores based on specific instrument configurations. Here is some information needed to get started:
| CellMarker | Large collection of manually curated information on cellular and tissue marker distribution and expression |
| AAI (American Association of Immunologists) | Resources to learn more about the immune system, form introductory to advanced |
| Web resources on basic immunology | Tutorials offered by academic institutions |
| Labome | Information on antigen density |
| Dye Database: | View the optical properties of more than one thousand fluorochromes and use the built-in spectra viewer to compare spectral signatures |
| Antibody Search | Extensive collection of over 3 million antibodies from all major suppliers |
Panel Design & Fluorophore Selection: to better navigate the complexity of panel design and access information about all the commercially available fluorescently-labeled antibodies, FluoroFinder has developed a platform that integrates our panel design tool with a comprehensive antibody and fluorophore database. Instrument-specific laser and filter settings allow the selection of compatible fluorophores. A spectra viewer lets users simultaneously view the optical properties, and emission and excitation profiles of the selected fluorophores to help minimize spectral overlap. The platform also offers the option to save, export, and share experiments, compare products, and request a quote from multiple suppliers. Return any time to optimize or clone prior experiments. Additional advanced functionalities include:
- Antigen density slider allows manual adjustment of antigen density so that only compatible fluorophores are displayed in each channel. Brighter fluorophores should be paired with low-expressed markers, while dimmer fluorophores are recommended when working with strongly expresses antigens to avoid spillover and reduce the need for complex and time-consuming compensation [1].
- Spectral panel design is possible due to the integration of all commercially available spectral cytometers configurations into the panel builder. Selecting a spectral instrument triggers the spectral panel design functionality and full spectral profiles of fluorophores can be viewed in the spectra viewer with accompanying similarity matrix data.
- OMIPS: FluoroFinder is developing a new OMIP (Optimized Multicolor Immunofluorescence Panel) [4] panel design functionality that integrated pre-loaded OMIPs panels and guides users through the panel building process.
Sample preparation
Sample preparation is a critical step in the experimental workflow. Accuracy and reproducibility are highly dependent on the choice of reagents and the methods used to preserve, isolate, fix, and stain the cells that will be analyzed. To quickly detect artifacts and identify potential problems it is essential to include the appropriate controls [2] [3]. Some useful information about sample preparation and experimental controls can be found here:
Tips and Tools for flow cytometry sample prep success
Instruments
FluoroFinder compiled an extensive list of cytometers and cell sorters to make it easy to compare the different instrument’s capabilities:
Analysis Software
A comprehensive list of commercially available and free software for flow cytometry analysis is available here
Networking opportunities
Check Fluorofinder’s events page to learn about opportunities to interact with the flow cytometry community, in person or virtually:
References:
[1] Roederer M. Compensation in flow cytometry. Curr Protoc Cytom.2002
[2] Hulspas R, O’Gorman MRG, Wood BL, Gratama JW, Sutherland DR. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom. 2009
[3] Keeney M, Gratama JW, Chin-Yee IH, Sutherland DR, Isotype controls in the analysis of lymphocytes and CD34+ stem and progenitor cells by flow cytometry – time to let go! Cytometry. 1998
[4] Yolanda Mahnke, Pratip Chattopadhyay, and Mario Roederer, Publication of optimized multicolor immunofluorescence panels, Cytometry, Part A. 2010
