Cytokine concentrations in supernatants from lung homogenates were quantified using the mouse BD Bioscience cytometric bead array following the manufacturer's instructions. Data were acquired using FACSCantoII and analysed using the flowjo software vs10 (Tree Star) and Prism 7 (graphpad software).
Facscanto 2
Manufactured by Tree Star
Sourced in United States
The FACSCanto II is a flow cytometry instrument designed for accurate cell analysis and sorting. It features advanced optics, detectors, and software to enable precise measurement and characterization of cell populations. The FACSCanto II provides core functionality for flow cytometry applications without interpretation or extrapolation.
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Lab products found in correlation
Anti igm, by Jackson ImmunoResearch (5 mentions)
Cd40l, by Enzo Life Sciences (5 mentions)
Il 21, by Thermo Fisher Scientific (5 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (4 mentions)
Streptavidin pe, by BD (4 mentions)
Isotype matched antibodies, by Thermo Fisher Scientific (3 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (3 mentions)
Annexin 5 fitc apoptosis detection kit, by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Facscaliber, by Tree Star (2 mentions)
Fc block 2.4g2, by BioXCell (2 mentions)
Pe conjugated anti ctla 4 uc10 4f10 11 pe cy7 conjugated anti cd25 pc81 biotin conjugated anti cd8b 53 5.8, by BioLegend (2 mentions)
Pacific blue conjugated anti cd4 rm4 5, by BioLegend (2 mentions)
Percp cy5.5 conjugated anti thy1 1 ox 7, by Thermo Fisher Scientific (2 mentions)
Brilliant violet421 conjugated anti human cd4 okt4, by Thermo Fisher Scientific (2 mentions)
Apc conjugated il 10 jess 16e3, by Cytek Biosciences (2 mentions)
Efluor660 conjugated anti gata 3 twaj, by Cytek Biosciences (2 mentions)
Fitc conjugated anti cd4 gk1.5, by Thermo Fisher Scientific (2 mentions)
R pe conjugated anti human cd4 s3.5, by Thermo Fisher Scientific (2 mentions)
Facs aria fusion, by Tree Star (2 mentions)
81 protocols using facscanto 2
1
Cytokine Quantification in Lung Homogenates
2
Isolation and flow cytometry of cecal cells
3
Multiparameter Phenotyping of Dendritic Cells and T Cells
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Directly conjugated mAbs against CD1c-BV421 (BD Biosciences), HLA-DR-PERCP (Invitrogen, Waltham, MA, USA), B7H3(CD276)-APC (Miltenyi, Stockholm, Sweden), B7H1-PE (Invitrogen), NOS1-FITC (Santa Cruz Biotechnology, Dallas, TX, USA), PDL2-APC (R&Dsystems), HVEM-PE (Invitrogen), Arginase 1-PerCP-eFluor 710 (Thermo fisher, Waltham, MA, USA), IDO-FITC (R&Dsystems), CD80-APC (Miltenyi), CD86-PE (BD Biosciences), CD85d (ILT4)-PerCP-eFluor 710 (Thermo fisher), COX2-FITC (Invitrogen), CD30L/TNFSF8-APC (R&Dsystems), Galectin-9-PE (Miltenyi), and B7-H4-Alexa Fluor 700 (R&Dsystems) were used for DC and T cell phenotyping before and after the DC–T cell interaction in the coculture. In brief the cells in the coculture were harvested, washed, and stained with the mAbs for 20-30 min at 4°C. Thereafter the cells were fixed with 4% PFA (Sigma Aldrich), permeabilized with 0.2% Saponin (Sigma Aldrich), and intracellular staining was performed at 4°C for 30-45 min. Data was acquired using FACS canto II, and the FlowJo software v9 (Treestar, OR, USA) was used for data analysis.
4
Flow Cytometry Analysis of T-cell Subsets
5
Intracellular ROS Measurement in RAW264.7 Cells
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RAW264.7 cells were preincubated with or without 4-PG (10 μM) for 6 h and N-acetyl-L-cysteine (NAC, 1 mM) for 30 min followed by the stimulation of LPS (100 ng/ml) for another 4 h. Then, medium was removed and cells were incubated in 1x PBS containing 5 μM of CM-H2DCFDA (C6827, Invitrogen) for 30 min at 37°C. The measurement of intracellular ROS was performed using a flow cytometer with fluorescence-activated cell sorter (FACSCanto II) and analyzed by FlowJo V10 software (Tree Star Inc., San Carlos, CA).
6
Comprehensive Immune Cell Analysis
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In order to analyze immune cells and their anti-tumoral activity, B6 mice were challenged and swarm at TT and BT (n=9 per group) and then 3 mice of them were randomly sacrificed every wk. Primary cells were collected from lymph node (LN), draining lymph node (dLN), and spleen, and analyzed via FACS Canto II or FACS Aria I. Data were analyzed using FlowJo version 10 (TreeStar, San Carlos, CA, USA). Antibodies with the following specificities were used for staining: CD8α (53-6.7), IFNγ (XMG1.2), IL-4 (11B11), CD44 (IM7), CD62L (MEL-14), NK1.1 (PK136), TCRβ (H57-597), and γδTCR (GL3) from BioLegend (San Jose, CA, USA); CD4 (GK1.5) from Thermo Fisher Scientific (Waltham, MA, USA); and IL-17 (TC11-18H10) from BD Biosciences (San Jose, CA, USA). Anti-mouse CD16/32 (2.4G2; BioLegend) blocked the non-specific binding of the antibodies. For intracellular cytokine staining, the cells were stimulated with PMA (Merck Millipore, Burlington, MA, USA) and ionomycin (Santa Cruz, Dallas, TX, USA) in the presence of brefeldin A (Thermo Fisher Scientific), which were then fixed and permeabilized with an IC fixation buffer (Thermo Fisher Scientific).
7
Analyzing Emergency Granulopoiesis in Mice
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Emergency granulopoiesis was analyzed according to a previous report (20 (link)). Briefly, WT, littermate WT, and FPR2 KO mice used for analysis were males from 7 to 9 weeks old. Cells were surface-stained in PEB buffer (PBS supplemented with and 2 mM EDTA and 0.5% BSA) for 30–45 min on ice. Antibodies used for flow cytometry included the following: Percp-Cy5.5-conjugated lineage markers specific for CD3e (Clone: 145-2C110), CD4 (Clone: RM4-5), CD8a (Clone: 53-6.7), CD11b (Clone: M1/70), B220 (Clone: RA3-6B2), GR-1 (Clone: RB6-8C5), and Ter119 (Clone: Ter119) from Thermo Fisher scientific (Waltham, MA, USA) or BioLegend (San Diego, CA, USA). Other antibodies included those against APC- or PE-conjugated Sca-1 (Clone: D7), c-kit (Clone: 2B8), FITC- or PE-Cy7-conjugated CD16/32 (Clone: 93), and CD34 (Clone: RAM34). Neutrophils were gated by CD11b+ and Ly6G+ (Clone: 1A8). Unstained cells were used as negative controls to establish flow cytometer voltage settings and single-color positive controls were used to adjust the compensation. Flow cytometric data were acquired using FACScantoII and analyzed with Flowjo software (Tree Star Inc. Ashand, OR, USA).
8
Irradiated Cell Surface Phenotyping
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Irradiated (100 Gy) and non-irradiated 4T1 and EMT6 cells (5 × 105) were stained with fluorochrome-conjugated anti-CD80 (clone 16-10A1), anti-CD86 (clone GL1), anti-H-2Kb (MHC I) (clone AF6–88.5), anti-I-Ad/I-Ed (MHC II) (clone M5/114.15.2), anti-CD54 (ICAM-1) (clone 3E2), and anti-CD95 (FasR) (clone Jo2) (BD Biosciences). Cells were analyzed on a FACSCantoII and differences in median fluorescence intensities (ΔMFI) between unstained and stained cells were determined using FlowJo software (Tree Star, San Carlos, CA, USA).
9
Multi-parameter Analysis of NK Cells
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All antibodies were purchased from BD Biosciences (San Jose, CA, USA) with the exception of phycoerythrin (PE)-conjugated anti-CD158a, anti-CD158b, anti-NKG2A, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46 and anti-TRAIL antibodies, which were purchased from R&D Systems (Minneapolis, MN, USA). NK subset frequencies and NK receptor expressions were analysed according to previously described protocols26 (link)50 (link). To detect NK cell activation, PBMCs were incubated with PE-conjugated anti-CD3, APC-conjugated anti-CD56 and PerCP-conjugated anti-HLA-DR, or FITC-conjugated anti-CD69 and anti-CD38. For intracellular staining, cells were permeabilised and stained with the corresponding intracellular antibodies, including those recognising perforin, granzyme A and B. Cells were then analysed using FACSCaliber or FACSCanto II and Flowjo software (TreeStar, Ashand, OR, USA).
10
Multiparametric Flow Cytometry Profiling
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Cells were stained at 4 °C in the presence of Fc Block (2.4G2; BioXcell) in flow cytometry buffer (0.5% BSA in PBS). The following antibodies were purchased from Becton Dickinson (BD): PE-conjugated anti-CTLA-4 (UC10-4F10-11); PE-Cy7–conjugated anti-CD25 (PC81); biotin-conjugated anti-CD8b (53-5.8), from BioLegend: Pacific blue-conjugated anti-CD4 (RM4-5); PerCP/Cy5.5–conjugated anti-Thy1.1 (OX-7); Brilliant Violet421–conjugated anti-human CD4 (OKT4); APC-conjugated anti-human CD4 (RPA-T4), biotin-conjugated anti CD45R/B220 (RA3-6B2), from eBioscience: APC-conjugated IL-10 (JESS-16E3); efluor660-conjugated anti-GATA-3 (TWAJ); PE-conjugated anti-IRF4 (3E4), biotin-conjugated anti-CD49b (DX5), from Tonbo Biosciences: FITC conjugated anti-CD4 (GK1.5), from Invitrogen, R-PE-conjugated anti-human CD4 (S3.5). For IL-10 and CTLA-4 staining, cells were fixed in 2% paraformaldehyde for 15 min at RT and permeabilized with 0.5% Saponin before staining. For BATF, GATA-3 and IRF4, cells were fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) following manufacturers’ instructions. Cells were analyzed on a FACSCanto II or FACS Aria Fusion and data were analyzed with FlowJo software (TreeStar).
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