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Department of Microbiology and Immunology

Guide to Rigor and Reproducibility for Flow Cytometry Experiments

You may use this as a template for wording in your grants and publications to address Rigor and Reproducibility for your Flow Cytometry Experiments run in the UNC Flow Cytometry Core Facility.

 

Eight steps to Rigorous and Reproducible Experiments in Biomolecular Research at UNC:

  1. If using a core facility, consult with the core staff in the planning stage. Consult with a statistician if you need help developing a Power Analysis to assure that your results will be adequately powered.

  2. Design your experiment with sufficient controls (rigor) and replicates (reproducibility).

  3. Assure that ALL of your reagents (antibodies, cell lines, mice) are fully validated (see below).

  4. Have a clear and detailed protocol (SOP) and data analysis plan that can be easily followed. Assure that the protocol is strictly followed or that any deviation is well documented.

  5. Assure that the staff or students performing the experiment are well trained and understand each step and the importance of performing them precisely (rigor again).

  6. Use only well-maintained instrumentation, preferably maintained and operated in a core facility with expert staff (see #1 above).

  7. Document all steps, reagents, equipment and data analysis methods used in the experiment. Assure that the both the documentation and the data itself are properly stored in a safe data management repository.

  8. Acknowledge the Cancer Center Support Grant (P30 CA016086), Grant-supported instrumentation and core staff in publications.

Consider Glenn Begley’s Six Rules for Reproducibility:

  1. Were the studies blinded?

  2. Were all the results shown?

  3. Were the experiments repeated?

  4. Were the positive and negative controls shown?

  5. Were the reagents validated?

  6. Were the statistical tests appropriate?

Guide to Rigor and Reproducibility for Flow Cytometry Experiments in the UNC Flow Cytometry Core Facility

I. Multicolor Flow Cytometry: all flow cytometry experiments will be run using replicate [animals/cell lines, etc] according to recommendations based on power calculations.

  • Include negative controls and single color controls with each experiment for sensitivity and compensation calculations.

  • Include a viability dye to exclude dead cells from the analysis

  • Incorporate Fluorescence Minus One (FMO) controls  to validate the expression of rare or low-expressing markers.

  • Run doublet discrimination in your analysis to exclude aggregates

  • Run time as a parameter to assure that the fluidics were running smoothly during acquisition

Daily quality control of all instrumentation is performed based on manufacturers’ recommendations to ascertain that all of the UNC Flow Cytometry Core Facility flow cytometers maintain peak performance. Flow cytometric fluorophore compensation and analysis is completed using FlowJo, Summit, or FCS Express.

II. Cell sorting: quality control/laser alignment and drop delay calculation is carried out according to manufacturers’ recommendations on all cell sorters prior to sorting. Single color controls are included with each experiment for compensation calculations. Post-sort analysis are run on each population to verify purity when possible.

III. All antibodies will be validated prior to reporting of results. Recommendations for research using antibodies (page 7-8) ‘Although vendor-supplied technical information may help investigators select reagents such as antibodies, this information is insufficient for validation’.

Antibody Validation: Standards, Policies and Practices Workshop Report 2016

Resources for antibody validation:

  1. Always Titrate

  2. Validate Specificity

  3. Integrate critical controls like Fluorescence Minus One controls (FMO)

  4. See: Uhlen et al., A Proposal for Validation of Antibodies, Nature Methods (2016)

  5. More at: https://expertcytometry.com/4-steps-to-validate-flow-cytometry-antibodies-and-improve-reproducibility/

  6. Also on the EuroMab network:

    Guidelines Website: https://www.euromabnet.com/guidelines/index.php   Guidelines Manuscript: https://www.ncbi.nlm.nih.gov/pubmed/26418356   Pos/Neg Controls Website: https://www.euromabnet.com/guidelines/positive-negative-controls.php   Additional Articles about Ab Validation: https://www.euromabnet.com/guidelines/articles-about-antibody-validation.php

As always, cite core support and appropriate instrumentation grants in your publications. This is important for center renewal!!

Learn about theNIH Initiative to Enhance Reproducibility through Rigor and Transparency. (Video)

Have your antibodies been validated? Check the Antibody registry.

Also, FluoroFinder has partnered with CiteAb to provide validation data for >200,000 reagents.

Resource Authentication Plan: https://grants.nih.gov/reproducibility/faqs.htm#V

What Kind of Information Should I Include in My Application’s Resource Authentication Plan? Check out instructions on NIH Nexus Blog.

What are ‘Key Biological and/or Chemical Resources’ that should be addressed your application’s authentication plan? Key biological and/or chemical resources include, but are not limited to, cell lines, specialty chemicals, antibodies and other biologics. More on NIH website

Explore Optimized Multicolor Immunofluorescence Panels (OMIPs) are peer-reviewed panels designed for fluorescent assays. Available via Fluorofinder.

Resources for preparing grant applications for NIH funding (examples and plans)

FASEB report on enhancing research reproducibility identifies three main gaps to research reproducibility:

  • Lack of uniform definitions to describe the problem

  • Insufficient reporting of key experimental details

  • Gaps in scientific training

Recommendations for research using antibodies (page 9-10) ‘Although vendor-supplied technical information may help investigators select reagents such as antibodies, this information is insufficient for validation’.