Experiment Overview

Repository ID: FR-FCM-Z2JV Experiment name: Phitonex 40 color MIFlowCyt score: 65.50%
Primary researcher: Seddon Thomas PI/manager: Michael Stadnisky Uploaded by: Michael Stadnisky
Experiment dates: 2020-02-17 - 2020-04-14 Dataset uploaded: Apr 2020 Last updated: Apr 2020
Keywords: [Innate Immune Response] [flow cytometry] [high content flow cytometry] [multicolor flow cytometry] [monocyte] [fluorescence] [CD4] [NK cells] [monocytes] [CD3+CD8+] [NKT cell] [Treg] [adaptive immunity] [Tregs] Manuscripts:
Organizations: Phitonex, Inc., Durham, North Carolina (United States)
Purpose: Taking a shared 35 color panel [1] we have used the 34 commercially available conjugated antibodies and incorporated our 6 new fluorescent labels. We aimed to assess the "plug and play" ability of 6 NovaFluor labels, as we have designed fluorescent labels that emit at targeted wavelengths with up to 70% less cross excitation than conventional tandem dyes.
Conclusion: We have added 6 colors with marginal impact on spread, except for the already difficult BB515-FITC combo. There was near-zero spread added to other channels.
Comments: For a deeper discussion of this data set and for all methods, please see our white paper: S.Y. Thomas, C. LaBoda, S. Burrows, D. Daley, A. Stroud, M.D. Stadnisky. "Above and Beyond 40 Colors." Phitonex, Inc. 2020. or email us info [at] Known issues: When you open the .wsp file in FlowJo, if you do not have the flowSOM plugin installed, you will get an error message. You do not need this to open the workspace, please select: 1. "OK" and then 2. "Use default" when prompted for the plugin folder location Data and analysis will also be posted to References: 1. Cytek Aurora: Say Hello to a New Reality. 2019.
Funding: Not disclosed
Quality control: Prior to the experiment, single color antibodies were titrated on human PBMCs to determine the optimal staining concentrations for cells. Live-Dead Blue Viability Dye was used to distinguish live cells. NovaBlock™ was used 5 minutes prior to antibody addition to block non-specific cell binding to labels. Single colors were generated on both cells and beads with the most optimal control (cells by default, beads if needed) used for unmixing raw data files. Using the raw data files in SpectroFlo, the full spectra for each label was compared to previously published Cytek spectra to make sure there was no mismatch. The Cytek Aurora instrument was calibrated to default settings prior to acquisition of data. Note: fcs files are either Aurora Raw or SpectroFlo Unmixed as labeled.
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