Single-cell suspensions were incubated with fluorochrome- or biotin-conjugated antibodies in the presence of anti-CD16/CD32 (Fc block, clone 2.4G2) as indicated. All samples were co-stained with a cell viability dye (Fixable dye eFluor780, Invitrogen). For cell sorting an iCyt Synergy (70-μm nozzle, Sony Biotechnology) was used. Intracellular cytokine staining was performed using BD Cytofix/Cytoperm Plus reagents (BD Biosciences) following pre-culture with RPMI, supplemented with 50 ng ml-1 phorbol 12-myristate 13-acetate (PMA), 500 ng ml-1 ionomycin and Protein Transport Inhibitor Cocktail (eBioscience), for 4 h at 37°C. Intracellular TF staining was performed using Foxp3 Staining kit reagents (eBioscience). Analysis was performed on an LSRFortessa system (BD Biosciences) with FACSDiva software (version 6.2, BD Biosciences). For cell sorting, an iCyt Synergy system (70-μm nozzle, Sony Biotechnology) was used. Data were analyzed with FlowJo software (version 10).
Icyt synergy
Manufactured by Sony
Sourced in United States, Switzerland
The ICyt Synergy is a versatile flow cytometer designed for multi-parameter analysis of cells and other particles. It features advanced optics, sensitive detectors, and powerful software for data acquisition and analysis. The core function of the ICyt Synergy is to provide researchers with a reliable and efficient tool for high-quality flow cytometry measurements.
Automatically generated - may contain errors
Lab products found in correlation
Winlist 3d, by Verity Software House (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa system, by BD (1 mentions)
Foxp3 staining kit reagents, by Thermo Fisher Scientific (1 mentions)
Protein transport inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
Collagenase 1, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Corn oil, by Merck Group (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Human fc blocking reagent, by Miltenyi Biotec (1 mentions)
Lsrfortessa, by BD (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
8 protocols using icyt synergy
1
Multiparameter Flow Cytometric Analysis
2
Isolation of Murine and Human Intestinal Macrophages
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Murine intestinal macrophages were flow-sorted as live CD11b+ CX3CR1+ Ly6Clo MHC-IIhi cells. Human ileal macrophages were flow-sorted as SSCint CX3CR1+ CD14+ cells. Cell sorting was performed on FACS Aria Fusion (BD biosciences), iCyt Synergy (Sony Biotechnology Inc.), and MoFlo (Beckman Coulter) cell sorters. Data were analyzed using FlowJo software (Tree Star Inc.).
3
Isolation of WAT-MSCs and WAT-ILC2s
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WAT was mechanically dissociated in RPMI-1640, and digested with collagenase I (Life Technologies) and DNase I (Roche) at 37°C while shaking. Lin–CD45–EpCAM–CD31–CD24–PDGFRα+CD34+Sca1+podoplanin+ WAT-MSCs and lineage–ST2+EpCAM– WAT-ILC2s were sorted by flow cytometry using Sony iCyt Synergy (Sony) to >95% purity. ILC2s were also isolated from mesenteric lymph node (MLN) as lineage–ST2+ cells. With the exception of the Matrigel study, all ILC2 and MSC assays were performed with cells isolated from naive C57BL/6 mice.
4
SLO-mediated cell permeabilization assay
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His-tagged SLO (carrying a cysteine deletion that eliminates the need for thiol activation26 (link)) was provided by R. Tweeten (University of Oklahoma, Norman, OK) and purified using Ni-nitrilo-triacetic acid (NTA) agarose resin (Qiagen) in BL21 Ecoli expression system. The protein concentration was measured by using the Bradford assay, and stored at −80 °C until use. For each experiment, about 2 × 106 cells were trypsinized, washed with Tyrode’s solution, and incubated with SLO (2–5 µg/ml) for 10 min at 37°C. Titration of SLO was performed to determine the minimum concentration required for cell permeabilization in 0 Ca2+ and the maximum concentration not causing significant cell loss. SLO-treated cells were then stained with 200 µg/ml propidium iodide (PI; Sigma-Aldrich) for 5 min and analyzed by FACS Flow Cytometry (iCyt Synergy; Sony). More than 10, 000 cells were used for each condition. Data were analyzed using WinList 3D software (Verity Software House).
5
SLO-mediated cell permeabilization assay
6
Tamoxifen-Induced CreTAM Activation
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CreTAM activation was induced by Tamoxifen administration in MD4 donor mice leading to the expression or deletion of proteins of interest. Tamoxifen (T-5648; 20 mg/ml final concentration; Sigma-Aldrich) was dissolved in corn oil (Sigma-Aldrich) containing 10% ethanol (Decon Laboratories). 100 µl (2 mg) was injected i.p. on two consecutive days. 6 d after the first Tamoxifen injection, spleens were harvested for subsequent experiments. At this time, deletion or expression of the proteins of interest was confirmed by immunoblotting or intracellular immunofluorescence. MD4 donor cells were stained with anti-B220–APC, and YFP+ B220+ events were sorted on a MoFlo XDP (Beckman Coulter) or an ICyt Synergy (Sony) cell sorter. 5 × 104–2 × 105 YFP+ B220+ cells were adoptively transferred by i.v. injection in 200 µl PBS at the indicated times after infection or in sham-infected mice or uninfected mice. 24 h after transfer, the mice were immunized by i.p. injection with 200 µl of 1% SRBC-HEL. 5 d later, serum and splenocytes were harvested and analyzed for anti-HEL responses.
7
PDL1 Expression Profiling by Flow Cytometry
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To preserve at least 300× library coverage, 30 million cells were seeded per treatment. Cells were treated with 10 ng/mL IFNγ (R&D Systems) for 72 h incubation, after which 30 million cells were incubated with a PDL1 monoclonal antibody (ThermoFisher #14-5983-82, 1 ug/mL in HBSS) for 15 min in a suspension of 1 million cells/mL followed by washing in PBS. Control (no primary antibody) and PDL1 stained cells were incubated with PE-conjugated rat anti-mouse secondary antibody (ThermoFisher #12-4015-82, 0.2 ug/mL in HBSS) for 15 min. Cell suspensions in HBSS supplemented with 0.5% FBS were gated on live cells and sorted on an iCyt Synergy (Sony Biotechnology, San Jose, CA, USA) at the University of Michigan Flow Cytometry Core.
8
Multiparametric Flow Cytometric Analysis of Immune Cells
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Cell suspensions were pre-incubated with 5 μL human Fc Blocking Reagent (Miltenyi Biotech, Bisley, UK) or normal rat serum (in dilution 1:100) for 10 minutes at 4 °C. Aqua Live/Dead™ 405 nm cell stain (Invitrogen, Paisley, UK) or Zombie UV Aqua Fixable Viability Stain (Biolegend, London, UK) was added in dilution 1:200 in PBS and incubated for 5 minutes, followed by the surface antibody cocktail for 30 mins in the dark at 4 °C. All rat (anti-mouse) and mouse (anti-human) antibodies were used in dilution 1:100 and 1:25, respectively. All antibodies used in this study are listed in Supplementary Table 1 . Intranuclear Ki-67 staining was performed using eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific, Waltham, USA).
Following three washes, samples were measured with BD LSRFortessa using BD FACSDiva 6.2 software (Becton Dickinson, Basel, Switzerland) and data subsequently analysed with FlowJo 10.4 software (TreeStar, USA). Each FACS tube was run until its exhaustion. Cell sorting of murine kidney and bladder MNPs and B cells was done on unfixed cells using Aria-Fusion III (Becton Dickinson, Basel, Switzerland) or iCyt Synergy (Sony Biotechnology Inc.).
Following three washes, samples were measured with BD LSRFortessa using BD FACSDiva 6.2 software (Becton Dickinson, Basel, Switzerland) and data subsequently analysed with FlowJo 10.4 software (TreeStar, USA). Each FACS tube was run until its exhaustion. Cell sorting of murine kidney and bladder MNPs and B cells was done on unfixed cells using Aria-Fusion III (Becton Dickinson, Basel, Switzerland) or iCyt Synergy (Sony Biotechnology Inc.).
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