For flow cytometry analysis and FACS, cells were dissociated using TrypLE Express, washed, and resuspended in FACS buffer (PBS with 10% FBS). For surface antigens, live cells were stained with fluorophore-conjugated antibodies and CellTrace Calcein Blue, AM (Life Technologies, C34853) at 1×107 cells/ml for 30 minutes on ice in the dark. For intracellular staining, cells were fixed using BD Cytofix fixation buffer (BD Biosciences, 554655) at 1×107 cells/ml for 20 minutes, washed with BD Perm/Wash buffer (BD Biosciences, 554723), and permeabilized in BD Perm/Wash buffer for 10 minutes, then stained with fluorophore-conjugated antibodies and DAPI in the dark for 30 minutes. Stained cells were washed twice with FACS buffer, filtered into a strainer-capped tube (Falcon, 352235), and run on a BD LSRFortessa. FACS was performed on a BD FACSAria or Beckman Coulter MoFlo Astrios. Spectral overlap was compensated using single fluorophore-conjugated AbC Total Antibody Compensation Beads (Life Technologies, A10497) with single fluorophore-conjugated antibodies. All antibodies are listed in Supplementary Table 6 . Flow cytometry data was analyzed using FlowJo 10.2.
Abc total antibody compensation beads
Manufactured by Thermo Fisher Scientific
The AbC Total Antibody Compensation Beads are designed for use in flow cytometry applications. The beads are coated with antibodies that can be used to compensate for spectral overlap between fluorescent dyes. This allows for accurate measurement and analysis of multiple fluorescent labels in a single sample.
Automatically generated - may contain errors
Lab products found in correlation
Perm wash buffer, by BD (2 mentions)
Moflo astrio, by Beckman Coulter (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cytofix fixation buffer, by BD (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Spectral flow cytometer, by Cytek (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
Cd45 apc cy7 clone 30 f11, by BD (1 mentions)
Cd4 bv421 clone gk1, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cd34 pe cy7, by BD (1 mentions)
Horizon brilliant stain buffer, by BD (1 mentions)
Facsdiva 8, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Zombie violet fixable viability stain, by BioLegend (1 mentions)
12 protocols using abc total antibody compensation beads
1
Comprehensive Flow Cytometry Analysis Protocol
2
Comprehensive Flow Cytometry Analysis Protocol
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For flow cytometry analysis and FACS, cells were dissociated using TrypLE Express, washed, and resuspended in FACS buffer (PBS with 10% FBS). For surface antigens, live cells were stained with fluorophore-conjugated antibodies and CellTrace Calcein Blue, AM (Life Technologies, C34853) at 1×107 cells/ml for 30 minutes on ice in the dark. For intracellular staining, cells were fixed using BD Cytofix fixation buffer (BD Biosciences, 554655) at 1×107 cells/ml for 20 minutes, washed with BD Perm/Wash buffer (BD Biosciences, 554723), and permeabilized in BD Perm/Wash buffer for 10 minutes, then stained with fluorophore-conjugated antibodies and DAPI in the dark for 30 minutes. Stained cells were washed twice with FACS buffer, filtered into a strainer-capped tube (Falcon, 352235), and run on a BD LSRFortessa. FACS was performed on a BD FACSAria or Beckman Coulter MoFlo Astrios. Spectral overlap was compensated using single fluorophore-conjugated AbC Total Antibody Compensation Beads (Life Technologies, A10497) with single fluorophore-conjugated antibodies. All antibodies are listed in Supplementary Table 6 . Flow cytometry data was analyzed using FlowJo 10.2.
3
Pneumococcal and GBS Polysaccharide Quantification
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One drop of AbC total antibody compensation beads (Thermo Fisher; A10513) was incubated with 40 μL of PS serotype-specific rabbit antisera (SSI Diagnostica) for 15 min at room temperature. The beads were then washed twice with FACS buffer (2 mM EDTA and 0.5% BSA in PBS) and spun for 5 min at 600×g. The antiserum-coupled beads were then incubated with 11.5 µg/mL (Spn) or 3.5 µg/mL (GBS) PS-SA multimers in FACS buffer for 15 min at room temperature (Spn) or on ice (GBS). Multimers were centrifuged prior to use at 2500 × g for 5 min at 4 °C to pull down aggregates that may cause artefacts in the staining. The beads were then washed two times with 1 mL FACS buffer, spun for 5 min at 600×g, resuspended in 300 μL FACS buffer and acquired with a spectral flow cytometer (Cytek) on the same day.
4
Isolation and Characterization of Murine PBMCs
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Blood was harvested from animals by submandibular bleed with a 5mm lancet. Peripheral blood mononuclear cells (PBMC) were isolated using gradient centrifugation with Ficoll-Paque Plus (Cytiva). Blood diluted 2:1 in RPMI was overlayed onto Ficoll-Paque and centrifuged for 15 minutes at 350g. The buffy coat was aspirated and washed with RPMI. PBMCs were stained with a panel of fluorochrome-conjugated antibodies reactive with the following cell surface markers: CD45-APC/Cy7 (clone 30-F11; BD Biosciences), CD3-PE/Cy7 (clone 500A2; Biolegend, San Diego, CA), CD8-FITC (clone KT15; ThermoFisher Scientific, Waltham, MA), CD4-BV421 (clone GK1.5; BD Biosciences). Antibody titrations are available in Supplemental Table 1 . PBMCs were also stained with Zombie Aqua (1:320 dilution; Biolegend, San Diego, CA) to identify dead cells. To identify T cells specific for mLama4 or mItgb1, PBMCs were stained with H-2K(b)-mLama4 (VGFNFRTL) or I-A(b)-mItgb1 (VNGYNEAIVHVVETP) MHC tetramers conjugated to PE and APC obtained from the NIH Tetramer Core Facility. Stained cells were acquired on a BD LSRFortessa Cell Analyzer at the Duke Human Vaccine Institute Flow Cytometry Core Facility. Compensation was accounted for using AbC Total Antibody Compensation Beads (ThermoFisher Scientific, Waltham, MA). Data were analyzed using FlowJo 10.
5
Hematopoietic Progenitor Cell Identification
6
Single Cell Sorting and RNA-seq
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AbC Total Antibody Compensation Beads (Thermo Fisher) were used to generate single color compensation controls prior to sorting. Sorting was conducted on either the FACSAria II, FACSAria IIu, or FACSAria Fusion instruments using a 70 μM nozzle, and cells were collected in 5 ml tubes pre-coated with FBS. A sample of each sorted cell population was reanalyzed on the same machine to assess purity. Cells were collected by centrifuging for 10 minutes at 500 g, and the cell pellet was frozen at −80°C for further processing. When sorting cells for RNA-seq, cells were collected in 5 ml tubes pre-coated with both FBS and RNAlater (Thermo Fisher).
7
Single Cell Sorting and RNA-seq
8
Characterizing MAIT Cells by Flow Cytometry
9
Antibody Spillover Compensation
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For the bead-based compensation of the signal spillover, AbC total antibody compensation beads (Thermo Fisher Scientific) were single stained with each of the antibodies used in Panel A and B according to manufacturer’s instructions.
10
Flow Cytometry Assay for moDCs
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For flow cytometry assays, moDCs were seeded at 100,000 cells per well in 100 µl of complete RPMI in a V-bottom 96-well plate. After incubation at hypoxic or atmospheric oxygen levels and with or without inhibitors, as described above, cells were centrifuged at 1500 rpm for 2 min at 4°C. Supernatant was discarded and cells were incubated for 30 min in the dark with PBS containing Zombie Violet fixable viability stain (BioLegend, 1:2000 dilution), then fixed with 4% PFA for 2 min and incubated for 10 min in the dark on ice with 50 µl of phosphate-buffered azide (PBA; PBS containing 0.5% BSA and 0.01% NaN3) containing 1% human serum to block Fc receptors. Then, cells were incubated for 30 min in the dark on ice with 50 µl of PBA containing directly labeled antibodies. To perform compensation and set gates, AbC Total Antibody Compensation beads (Thermo Fischer Scientific) were stained in the same way as the cell samples. Cells were washed with 100 µl PBA, resuspended in 60 µl PBA, and analyzed on a FACSCalibur or FACSVerse flow cytometer (BD Biosciences).
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