Single-cell suspensions in staining buffer (Biolegend) were incubated with anti-hEpCAM (CD326)-APC-Vio770 (1:100, Miltenyi Biotec), anti-mCD45-PE-Cy7 (1:300, Biolegend), anti-hCD44v6-PE (1:20, R&D System), and/or anti-mEpCAM-APC-Vio770 antibodies for 30 min on ice. After the cells were washed twice with 1 ml of the labeling buffer, they were analyzed on a FortessaX20 Flow Cytometer (BD Biosciences) or were sorted by MoFlo Astrios EQ Cell Sorter (Backman Coulter). For analysis of Nanog expression in tumor epithelial cells, single-cell suspensions were stained with cell surface markers as described above. Then, the cells were fixed and permeabilized by using a Cytofix/Cytoperm kit (BD Biosciences) followed by intracellular cellular staining with primary anti-human Nanog rabbit antibody (1:100, abcam) in permeabilization buffer for 30 min on ice. After washing with permeabilization buffer, the cells were incubated with second anti-rabbit-IgG-PE (1:200, Biolegend) in permeabilization buffer for 30 min on ice. After the cells were washed twice with 1 ml of the permeabilization buffer, they were analyzed on a FortessaX20 Flow Cytometer (BD Biosciences). The flow cytometric profiles were analyzed by counting 20,000 events using FLOWJO software program (FLOWJO, LLC).
Fortessa x20 flow cytometer
Manufactured by BD
Sourced in United States, Netherlands
The Fortessa X20 is a flow cytometer designed for high-performance cell analysis. It features a compact design and offers multi-color detection capabilities for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (4 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (4 mentions)
Cytofix cytoperm, by BD (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Kaluza 2, by Beckman Coulter (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (2 mentions)
Influx cell sorter, by BD (2 mentions)
Apc conjugated anti human fc, by Jackson ImmunoResearch (2 mentions)
Fitc labeled secondary antibody, by Jackson ImmunoResearch (2 mentions)
Golgiplug, by BD (2 mentions)
Zombie aqua fixable viability dye, by BioLegend (2 mentions)
Anti cd206 pe, by BioLegend (2 mentions)
Anti cd11b percp cy5, by BD (2 mentions)
Siglecf pe, by BD (2 mentions)
Anti c kit apc, by BD (2 mentions)
F4 80 apc, by BD (2 mentions)
Fc block, by BD (2 mentions)
Anti ly6g fitc, by BD (2 mentions)
Apc labeled secondary antibody, by Jackson ImmunoResearch (2 mentions)
111 protocols using fortessa x20 flow cytometer
1
Multi-marker flow cytometry analysis of tumor cells
2
Flow Cytometric Analysis of Protein Expression
3
Flow Cytometric Analysis of Liposomes
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Cell suspensions were analyzed with a FORTESSA X20 flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA). The extracellular fluorescence of the NBD-PE lipid present in the liposomes or the FITC conjugated to the nucleic acid was quenched using trypan blue as in [23 (link),44 (link),45 (link)]. The cell-associated fluorescence was then measured with a flow cytometer (FACSort; Becton Dickinson, Franklin Lakes, New Jersey) with λex = 488 nm; λem = 530 ± 30 nm. The fluorescence intensity was expressed as the mean fluorescence intensity of 10,000 events.
4
Comprehensive CAR T Cell Analysis
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Antigen expression was detected using 5 μg/mL of GCT02 IgG (ATUM, Newark, USA) or 5 μg mL−1 of Cetuximab (Erbitux, Merck Serono, Darmstadt, Germany) and mouse anti‐human IgG‐BV711 (Becton Dickenson Biosciences, New Jersey, USA) secondary. To determine CAR cell surface expression, αMYCtag or αFLAG‐tag, αCD3, αCD4, αCD8 antibodies were used (refer to Supplementary table 1 ). For phenotyping, T cells were labelled with Fixable Yellow (Invitrogen, Waltham, USA), αCD45, αCD4, αCD8, αCD45RO and αCD197 antibodies (Supplementary table 1 ). CAR T cell degranulation was measured by coculture of three independent PBMC donors at a 1:1 ratio with αCD3/CD28 Dynabeads® (Life Technologies, Carlsbad, USA), U118/EGFRvIII, primary keratinocytes or astrocytes and the αCD107a‐PE antibody (Becton Dickenson Pharmingen) at 37°C, 5% CO2 for 4 h. Cells were labelled with a αCD8 antibody (Supplementary table 1 ), before washing and adding DAPI before flow cytometry analysis, using a Becton Dickenson FortessaX20 flow cytometer and FlowJo software (Becton Dickenson, version 10.8).
5
Isolation and Characterization of Mouse Mammary Epithelial Subpopulations
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Primary mouse mammary gland cells were used as controls for flow cytometry and were isolated using mechanical and enzymatic disaggregation as described previously [37 (link)]. The epithelial subpopulations were isolated from the disaggregated samples by flow cytometry as detailed previously [18 (link),19 (link),38 (link)]. Hematopoietic lineage cells were stained with anti-CD45-PE-Cy7, anti-TER119-PE-Cy7 and anti-CD31-PE-Cy7 (BD Biosciences, Franklin Lakes, NJ, USA). Cells were simultaneously stained with mammary-specific lineage markers, anti-Epcam BV421(BD Biosciences), anti-CD49f-APC (Biolegend, San Diego, CA, USA), anti-Sca-1-PE (BD Biosciences), anti-CD49b-FITC (Biolegend, San Diego, CA, USA) and propidium iodide (Sigma). Cells were analysed on a Fortessa-X20 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Linear density contour plots were used to describe flow cytometry gates. Fluorescence-minus-one control gates defined marker-negative populations.
6
Evaluation of Splenic DC Transfection
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Specific pathogen-free female BALB/c (8–11 weeks) mice were obtained from Janvier (Le Genest St Isle, France) and kept in isolated ventilated cages. All animal studies were approved by the French Ministry of Agriculture for experiments with laboratory animals.
Mice received 20 μg EGFP mRNA/LPP in HEPES 5% sucrose by i.v. injection in the tail vein (volume of 250 μL). Control mice received 250 μL HEPES 5% sucrose. Evaluation of splenic DC transfection was carried out as described in Perche et al.7 Spleens were harvested 24 h or 48 h after injection. Splenic cells were isolated and labeled with magnetic anti-mouse CD11c antibodies (Miltenyi Biotec SAS, Paris, France), which bound to DCs and were enriched by immunomagnetic selection using MACS MS columns (Miltenyi Biotec SAS) according to the manufacturer’s protocol. Then, cells were stained with BV711-labeled anti-mouse CD11c antibodies (Miltenyi Biotec SAS), and at least 40,000 cells were analyzed with a FORTESSA X20 flow cytometer (Becton Dickinson) with λex = 488 nm, λem = 530/30 nm for EGFP fluorescence and λex = 405 nm, λem = 710/50 nm for BV711 fluorescence. BV711 is a Horizon Brilliant Violet tandem fluorophore (Beckton Dickinson) of BD Horizon BV421 with a maximum λex at 405 nm and an acceptor dye with a maximum λem at 711 nm.
Mice received 20 μg EGFP mRNA/LPP in HEPES 5% sucrose by i.v. injection in the tail vein (volume of 250 μL). Control mice received 250 μL HEPES 5% sucrose. Evaluation of splenic DC transfection was carried out as described in Perche et al.7 Spleens were harvested 24 h or 48 h after injection. Splenic cells were isolated and labeled with magnetic anti-mouse CD11c antibodies (Miltenyi Biotec SAS, Paris, France), which bound to DCs and were enriched by immunomagnetic selection using MACS MS columns (Miltenyi Biotec SAS) according to the manufacturer’s protocol. Then, cells were stained with BV711-labeled anti-mouse CD11c antibodies (Miltenyi Biotec SAS), and at least 40,000 cells were analyzed with a FORTESSA X20 flow cytometer (Becton Dickinson) with λex = 488 nm, λem = 530/30 nm for EGFP fluorescence and λex = 405 nm, λem = 710/50 nm for BV711 fluorescence. BV711 is a Horizon Brilliant Violet tandem fluorophore (Beckton Dickinson) of BD Horizon BV421 with a maximum λex at 405 nm and an acceptor dye with a maximum λem at 711 nm.
7
Multiparametric Flow Cytometry for Treg Profiling
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Eleven-color flow cytometry was used to identify Tregs and the various Treg subsets. In brief, PBMCs were washed twice in phosphate-buffered saline, and then stained (30 minutes on ice) with Brilliant violet (BV) 711 anti-human CD3 (Clone UCHT1; Biolegend, San Diego, CA, USA), BV510 anti-human CD4 (Clone SK3, Biolegend), BV788 anti-human CD25 (Clone MA251; BD Biosciences, San Jose, CA, USA), BV650 anti-human CD45RA (Clone HI100, Biolegend), and with the following chemokine receptors: BV605 labeled CCR6 (Clone G04E3, Biolegend) and BV421 labeled CCR7 (Clone G043H7, Biolegend). After staining for surface molecules, intracellular staining was performed with allophycocyanin labeled mAB against FoxP3 (Clone PCH101; eBioscience, San Diego, CA, USA) and PeCF594 mAB against cytotoxic T-lymphocyte-associated protein (CTLA) 4 (Clone BN13, BD Biosciences), using a fixation and permeabilization solution (eBioscience) according to the manufacturer’s instructions. Then the cells were washed twice and analyzed with a Fortessa X20 flow cytometer (Becton Dickinson, San Jose, CA, USA) using FACS Diva V8.0 software (Becton Dickinson).
8
Chemokine Profiling of Mouse Tissues
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Individual tissue samples were collected in 0.5 mL of non-denaturing × 4.7 conc lysis buffer (Cell Signaling Technologies), with 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) added immediately prior to harvesting. Tissues were homogenized as described for RT-qPCR. Homogenates were clarified at 10,000×g for 5 min at 4 °C and frozen at − 80 °C. Mouse proinflammatory chemokines LEGENDplex assay (Biolegend) was performed with twofold-diluted samples. Data acquisition was performed using a FORTESSA X20 flow cytometer (Becton Dickinson) to acquire 4 000 beads/sample.
9
Apoptotic T Cell Quantification
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Whole blood was incubated with CD3 BV421 (Becton Dickinson). Erythrocytes were then lysed twice using Easylyse (Dako, Glostrup, Denmark). To evaluate the proportion of apoptotic T cells, PE-conjugated active caspase-3 apoptosis kit (Becton Dickinson) has been used. Samples were run in a Fortessa X20 flow cytometer (Becton Dickinson) and data were analyzed using Kaluza 2.0 software (Beckman Coulter).
10
Isolation and Characterization of Stromal Vascular Fraction from Adipose Tissue
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For stromal vascular fraction (SVF) isolation, eWAT of LFD- and HFD-fed mice was isolated and immediately rinsed in cold Phosphate Buffered Saline (PBS; Life Technologies, Carlsbad , USA). eWAT was then cut in 1-2 mm pieces and digested in Dulbecco modified Eagle's minimal essential medium (DMEM; Life Technologies) supplemented with 0.1% collagenase type I (C2674; Sigma-Aldrich, Saint-Louis, USA) at 37°C for 1h under agitation. Enriched-mature adipocyte cell fraction (floating cells) and SVF cells (pellet) were separated by centrifugation. Immune cells from SVF were characterized by flow cytometry using fluorochrome-conjugated surface protein-specific antibodies (Supp. Figure 2 ). Macrophages were stained using anti-CD45-BV510 (BLE103138; Biolegend, San Diego, USA), anti-CD11b-BUV395 (563553; BD Biosciences, San Jose, USA), anti-F4/80-PE/Cy7 (BLE123114; Biolegend), anti-CD11c-Percp-Cy5.5 (BLE117328; Biolegend) and anti-CD206-AF647 (BLE141712; Biolegend) antibodies. Cells were analysed using a FORTESSA X20 flow cytometer (Becton Dickinson, New Jersey, USA). Results were acquired using the FACS Diva software (BD Biosciences). Macrophages (CD45+ CD11b+ F4/80+) from FXR−/− Ad-FXR−/− and littermate mice were isolated using a BD Influx cell sorter (BD Biosciences).
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