The single gonadal cells were immunostained by SSEA-1 antibody (1:500 dilution) for 20 min on ice, followed by anti-mouse IgM conjugated by AF654 (1:5000 dilution) for 15 min on ice. The resulted staining was analysed by B&D Fortessa and FlowJo v10 software.
Fortessa
Manufactured by FlowJo
The Fortessa is a compact and versatile flow cytometer designed for a wide range of applications in life science research. It features advanced optics and electronics to deliver high-quality data, and is capable of detecting and analyzing multiple fluorescent parameters simultaneously. The Fortessa is a robust and reliable instrument that can be utilized across various research fields.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by FlowJo (2 mentions)
Live dead violet, by Thermo Fisher Scientific (1 mentions)
Microcentrifuge tube, by Eppendorf (1 mentions)
Mouse fc block, by BD (1 mentions)
Rat anti cd45 apc cy7, by BioLegend (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lsr 2, by BD (1 mentions)
Rnase a, by Merck Group (1 mentions)
Collagenase type a, by Roche (1 mentions)
Egm 2, by Lonza (1 mentions)
Ebm 2, by Lonza (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd8 pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Mhcii, by Thermo Fisher Scientific (1 mentions)
Anti cd44 percp cy5, by Thermo Fisher Scientific (1 mentions)
Anti il 2 apc, by Thermo Fisher Scientific (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
11 protocols using fortessa
1
Single Cell Immunostaining and Flow Cytometry
2
Cell Surface Staining and Bispecific mAb Binding
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For cell surface staining, cells were incubated with appropriate mAbs for 30 min on ice, washed, and incubated with secondary antibody reagents when necessary. For Foxp3-#32-bispecific mAb staining, human T cells or cancer cells were incubated with different concentrations of Foxp3-#32-bispecific mAb or control bispecific mAb for 30 min on ice, washed, and incubated with secondary mAb against His-Tag. Flow cytometry data were collected on a Beckman Dickinson Fortessa and analyzed with FlowJo 9.8.1 and FlowJo10 software.
3
Isolation and Staining of Microglia
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Suspensions of white blood cells and microglia were transferred to 2 ml Eppendorf microcentrifuge tubes, and a small fraction of sample was removed for single-color controls. To stain dead cells, live/dead violet (1:500–1:1000; ThermoFisher, L34955) was added and incubated for 5 min on ice. Tubes with live/dead-stained cells were topped off to 2 ml with FACS buffer (0.5% BSA HBSS, 1 mm EDTA) and spun down at 300 × g for 5 min at 4°C. Pellets were resuspended and incubated with 1:200 Mouse Fc Block (BD, 2.4G2, 553142) on ice for 15 min. Samples were incubated with 1:200 rat anti CD45-APC/Cy7 (BioLegend, 103115) and rat anti CD11b-PE/Cy5 (BioLegend, 101209) at 4°C. Tubes were topped off to 2 ml with ice-cold FACS buffer and microglia pelleted at 300 × g for 5 min at 4°C. Supernatants were removed and microglia resuspended in 500 μl. Resuspended microglia were filtered through corning strainer polystyrene tubes (Corning, 352235). Flow cytometry data were acquired on Aria II, Fortessa HTS, and LSRII HTS, and analyzed using FlowJo.
4
Fluorescence-based Cell Gating
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Gates were determined by fluorescence minus one (FMO) controls. Cells were acquired using Becton Dickinson Fortessa or FACSCanto II flow cytometers and data analysed using FlowJo Version 8.7.
5
Characterization of Tumor-Draining Lymph Node Cells
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After mechanical dissociation of tumor-draining lymph nodes, cells were stained with various mAbs including CD3, CD8α, CD11c, MHC class II, CD40, CD80, and CD86 and with fixable viability dye. After washing, cells were analyzed with an LSR Fortessa instrument and FlowJo software. The Abs were used according to the manufacturer’s instructions.
6
Cell Cycle Analysis by Flow Cytometry
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Single cell suspensions were fixed and permeabilized with 70% ice-cold ethanol. Next, cells were treated with 100 μg/ml RNaseA (Sigma) and stained with 10 μg/ml propidium iodide (Sigma). Data were collected using a BD LSRII and Fortessa flow cytometers and analyzed using FlowJo software.
7
HUVEC Isolation and Characterization
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Endothelial cells were harvested from human umbilical cords following written informed consent of the patients approved by the cantonal ethics committee Zurich, Switzerland (KEK-ZH-2009-0095). Briefly, HUVEC were dissociated from the venous lumen by incubating a collagenase solution (2 mg/ml collagenase type A, Roche Diagnostics GmBH, Switzerland) for 30 min in a humidified incubator at 37 °C and obtained in endothelial growth medium (EGM-2; Lonza, Switzerland).
After several passages, HUVEC were starved in endothelial basal medium (EBM-2; Lonza, Switzerland) with 1% FCS for 4 h and co-incubated for 16 h with either (I) hMSC CM + 0.5% FCS (n = 5 per hMSC group), (II) EGM-2 (n = 5) or (III) basal DMEM + 0.5% FCS (n = 5). 0.2 ml hMSC CM diluted 1:3 in basal DMEM was used per 100,000 HUVEC. Harvested HUVEC were stained 20 min at 4 °C with the following surface antibodies CD31, CD105, CD144, and CD202b (supplementary TableS2 ). 50,000 events acquired on a LSR Fortessa were analyzed by FlowJo software gating viable and single cells (for gating strategy see supplementary Fig. S13 ). Bidimensional t-SNE maps were generated (5000 events) and PhenoGraph algorithm applied.
After several passages, HUVEC were starved in endothelial basal medium (EBM-2; Lonza, Switzerland) with 1% FCS for 4 h and co-incubated for 16 h with either (I) hMSC CM + 0.5% FCS (n = 5 per hMSC group), (II) EGM-2 (n = 5) or (III) basal DMEM + 0.5% FCS (n = 5). 0.2 ml hMSC CM diluted 1:3 in basal DMEM was used per 100,000 HUVEC. Harvested HUVEC were stained 20 min at 4 °C with the following surface antibodies CD31, CD105, CD144, and CD202b (supplementary Table
8
Multiparametric Flow Cytometry Analysis
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Cells were harvested and following incubation with Fc block (homemade containing 24G2 supernatant and mouse serum) surface stained with anti‐CD4 APC‐Alexa 780 (eBioscience; clone: RM4‐5), anti‐CD44 PerCP‐Cy5.5 (eBioscience; clone: IM7), CD8 PeCy7 (eBioscience; clone: 53–6.7), and “dump” antibodies: B220 (clone: RA3‐6B2) and MHC II (clone: M5114) both on eFluor‐450 (eBioscience) for 20 min at 4°C. Cells were stained with a fixable viability dye eFluor 506 (eBioscience) as per the manufacturer's recommendations. Cells were fixed with cytofix/cytoperm (BD Bioscience) for 20 min at 4°C and stained in permwash buffer with anticytokine antibodies for 1 h at room temperature (anti‐IFN‐γ PE (clone: XMG1.2;), anti‐TNF Alexa‐Fluor‐488 (clone: MP6‐XT22), anti‐IL‐2 APC (clone: JES6‐5H4) all from eBioscience. Following washing with permwash buffer, samples were acquired on a BD LSR or Fortessa and analyzed using FlowJo (version 10 Treestar). Data are presented as required for MIFlowCyt.
9
Comprehensive Th Cell Phenotyping
10
Flow Cytometry Gating Analysis
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Gates were determined by fluorescence minus one (FMO) controls. Cells were acquired using Becton Dickinson Fortessa or FACSCanto II flow cytometers, and data analysed using FlowJo Version 8.7.
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