Rigor & Reproducibility
Department of Pediatrics Flow Cytometry Facility Guide to Rigor and Reproducibility
- Enhancing Research Reproducibility: Recommendations from the Federation of American Societies for Experimental Biology.
- For all Multi-color flow cytometry experiments users should run replicates based on Power Calculations. When doing multiple color experiments single color controls need to be included for calculating correct compensations (beads or cells can be used). If you have rare or low-expressing antigens then Fluorescence Minus One controls should be used to validate expression levels. All of the cores instrumentation with have QC performed daily according to the manufacturer’s recommendations. Core facility staff will ensure all instruments are running to manufacturers specification. Flow Cytometric Analysis and compensation will be performed using the following software: FlowJo, FCS Express 6, Kaluza or Cytxpert.
- Cell Sorting Assisted (FACS Aria II): facility staff will perform all QC, Daily Alignment (when required) as well as calculating the drop delay before each sort. A test sort using beads will also be performed to ensure the instrument is performing correctly. Purity checks or post sort analysis will be run whenever possible.
- Cell Sorting Unassisted (Sony Cell Sorter): Users will be responsible for performing correct startup procedure including following all wizards for QC and instruments setup/optimization. Purity checks or post sort analysis will be run whenever possible.
- Validation of Antibodies: Prior to reporting or publishing any results all antibodies should be validated. The table below has some great tips and links for validation:
- Make sure you validate your antibodies specificity.
- Titration of antibodies is essential.
- Use appropriate controls such as FMO’s and also positive controls if required.
- Expert Cytometry Antibody Validation
- Euromab Guidlines
- Euromab Positive and Negatives
- The European antibody network's practical guide to finding and validating suitable antibodies for research.
Check your Antibodies Validation using the Antibody Registry.
Optimized Multicolor Immunofluorescence Panels (OMIPs) are peer-reviewed panels designed for fluorescent assays. FluoroFinder can guide you through the process of building an OMIP in 3 steps as well as suggest alternative fluorochromes.