Flow cytometry was performed on either BD LSR-II or BD LSR Fortessa X-20 (BD Biosciences) cytometers at the UVRI-IAVI laboratory or UofT, respectively. Compensation was performed regularly using single stained compensation beads (BD Biosciences). The fluorescence output of the cytometer channels across the runs was standardized using Ultra rainbow calibration particles (Spherotech, USA). Analysis was performed using FlowJo software v.10.4.1 (TreeStar) with blinded participant/study visit identifications. Gating was guided by fluorescence minus 0 (FMO) controls. Cervical samples were excluded from analysis if the CD4+ T cell count per FCS file was less than 15 cells and/or if the control well appeared contaminated by pseudovirus.
Ultra rainbow calibration particles
Manufactured by Spherotech
Sourced in United States
The Ultra Rainbow Calibration Particles are a diverse set of fluorescent particles designed for instrument calibration and performance verification. These particles exhibit a broad spectrum of colors, allowing for comprehensive testing and validation of various imaging and detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii cytometer, by BD (2 mentions)
Anti pd1 phycoerythrin pe conjugated antibodies, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Single stained compensation beads, by BD (1 mentions)
Zombie uv fixable viability dye, by BioLegend (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Cytometer setup and tracking beads, by BD (1 mentions)
Stabilizing fixative, by BD (1 mentions)
Mhcii, by Thermo Fisher Scientific (1 mentions)
F4 80, by BD (1 mentions)
Anti cd16 32, by BD (1 mentions)
Siglec f, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Prism for mac, by GraphPad (1 mentions)
Cytometer setup and tracking beads cs t beads, by BD (1 mentions)
Rainbow calibration particles, by Spherotech (1 mentions)
Lsr fortessa special order research product, by BD (1 mentions)
8 protocols using ultra rainbow calibration particles
1
Standardizing Flow Cytometry Analysis
2
Flow Cytometric Immunophenotyping of Leukocytes
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Leukocyte viability was determined by staining with Zombie UV Fixable Viability Dye (BioLegend, San Diego, CA). Cells were incubated for 5 min at 4 °C with anti-CD16/32 (Becton Dickinson; BD, San Jose, CA, 2.4G2), then stained with the following antimouse antibodies: CD45.1 (Thermo Fisher, A20), CD45.2 (Thermo Fisher, 104), CD11b (Thermo Fisher, M1/70), CD11c (BD, HL3), Ly6C (BioLegend, HK1.4), Ly6G (BD, 1A8), F4/80 (BD, T45-2342), B220 (Thermo Fisher, RA3-6B2), NKp46 (BD, 29A1.4), CD3 (Thermo Fisher, 17A2), MHCII (Thermo Fisher, M5/115.15.2), and Siglec-F (BD, E50-2440). Cells were fixed with stabilizing fixative (BD), and data were acquired on a BD Special Order Research Product LSRFortessa flow cytometer. Quality control checks with Cytometer Setup and Tracking beads (BD) and Ultra Rainbow Calibration Particles (Spherotech, Lake Forest, IL) were performed before each acquisition. Data were cleaned using FlowAI32 (link) and analyzed using Flowjo software (BD).
3
Flow Cytometry Protocol for Cell Analysis
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Flow cytometry was performed on a FACSCanto II (BD Biosciences) using FACSDiva software (BD Biosciences). Before sample analysis, the flow cytometer settings were checked using Cytometer Setup and Tracking beads (CS&T beads, BD) according to the manufacturer’s instructions. Compensation beads were used with single stains of each antibody in order to determine the compensation settings, and these were applied in FlowJo software (version 10.0.6, Tree Star, Ashland, OR, USA) after data collection. The same compensation matrix was applied to all samples. A side scatter (SSC) threshold level was set at 4,000 units to eliminate debris. Gain settings were optimised for detection of stained populations using Ultra Rainbow Calibration Particles (SpheroTech Inc., Lake Forest IL, USA), and kept consistent throughout the study.
Recorded data were compensated post-hoc and analysed using FlowJo software. Statistical tests were performed in SPSS (IBM) and Prism for Mac (version 5, GraphPad, La Jolla CA, USA). Specific antibody staining data were visualised using bi-exponential transformation in FlowJo software. Gates used to discriminate positive and negative staining cells in FlowJo were set according to fluorescence minus one (FMO) tests of milk and blood samples, and these gates were applied consistently to all samples, allowing for minor adjustments for SSC variability.
Recorded data were compensated post-hoc and analysed using FlowJo software. Statistical tests were performed in SPSS (IBM) and Prism for Mac (version 5, GraphPad, La Jolla CA, USA). Specific antibody staining data were visualised using bi-exponential transformation in FlowJo software. Gates used to discriminate positive and negative staining cells in FlowJo were set according to fluorescence minus one (FMO) tests of milk and blood samples, and these gates were applied consistently to all samples, allowing for minor adjustments for SSC variability.
4
Expression of T Cell Co-Stimulatory Molecules
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Fresh live PBMCs were isolated over a Ficoll-Hypaque gradient and stained with anti-CD4 allophycocyanin cyanine (APC-Cy7), anti-ICOS allophycocyanin (APC), anti-CD28 peridinin chlorophyll protein cyanine (PerCP-Cy5.5), anti-CD45RO fluorescein isothiocyanate (FITC), and anti-PD1 phycoerythrin (PE) conjugated antibodies (Biolegend, eBiosciences) to determine the expression of co-stimulatory molecules on the CD4 T cell surface. Cells were analyzed on a LSR-II cytometer (Becton-Dickinson). Prior to each run, laser voltages were adjusted with Ultra Rainbow Calibration Particles (Spherotech) to ensure run-to-run consistency of the cytometer over the duration of this study. Data was analyzed using FlowJo software.
5
Expression of T Cell Co-Stimulatory Molecules
6
Conversion of MFI to MEFLs
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As shown in Supplementary Fig. 16 , to determine the conversion factor for MFI to MEFLs, Rainbow Calibration Particles (Spherotech, RCP-30-5) or UltraRainbow Calibration Particles (Spherotech URCP-100-2H) were run with each flow cytometry experiment. This reagent contains six (RCP) or nine (URCP) subpopulations of beads, each of a specific size and with a known number of various fluorophores. The total bead population was identified by SSC vs. FSC gating, and the subpopulations were identified through two fluorescent channels. The MEFL values corresponding to each subpopulation were supplied by the manufacturer. A calibration curve was generated for the experimentally determined MFI vs. manufacturer-supplied MEFLs, and a linear regression was performed with the constraint that 0 MFI equals 0 MEFLs. The slope from the regression was used as the conversion factor, and error was propagated.
7
Multicolor Flow Cytometry Analysis
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Flow cytometry was performed on a BD LSR Fortessa Special Order Research Product using the 562 nm laser for dTomato (582/15 filter), the 488 nm laser for EYFP (530/30 filter), and the 488 nm and 405 nm lasers for mTFP1 (530/30 filter and 525/50 filter, respectively). Approximately 10,000 live cells were collected per sample for analysis. Data were analyzed using FlowJo v10 (FlowJo, LLC). Briefly, cells were identified using an FSC-A vs SSC-A plot and gated for singlets using an FSC-A vs FSC-H plot (Supplementary Fig. 19). In transfection experiments, transfected cells were identified by fluorescence attributable to the transfection control by gating using a non-fluorescent control (Supplementary Fig. 20). Fluorescence data were compensated for spectral bleed-through where appropriate. Mean fluorescence intensity (MFI) of single-cell samples was exported and averaged across three biological replicates. Autofluorescence from untreated cells was subtracted from other samples. Standard error of the mean was propagated through calculations. Where indicated, 9 peak Ultra Rainbow Calibration Particles (Spherotech URCP-100-2H) were used to generate a calibration curve to convert fluorescence into absolute fluorescence units.
8
Quantifying Fluorescent Reporter Signals
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Flow cytometry data were analyzed using FlowJo software (FlowJo, LLC) to gate on single-cell (FSC-A versus FSC-H) and live (FSC-A versus SSC-A) bases, compensated using compensation control samples, and gated as transfection-positive (fig. S1A). The mean reporter signal in mean fluorescence intensity (MFI) was obtained for each sample. Ultra Rainbow Calibration Particles (Spherotech, #URCP-100-2H) were run in each flow cytometry experiment. Beads were gated on an FSC-A versus FSC-H basis, the nine bead subpopulations of varying intensities were identified, and the MFI for each subpopulation in the FITC channel and PE–Texas Red channel was obtained. These values in combination with manufacturer-supplied MEFL and MEPTR values for each subpopulation were used to fit a regression line with y intercept equal to zero. The mean and SEM for the three biological replicates were calculated. Autofluorescence background signal was subtracted using samples transfected with the transfection control marker, and error was propagated. MFI values were converted to MEFL or MEPTR using the slope of the regression line, and error was propagated. Histograms in supplementary figures represent reporter signal in MFI.
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