The supernatant of FBs stimulated by arecoline was used as the conditioned medium for the experimental group whereas the supernatant of FBs without arecoline stimulation was utilized as the conditioned medium for the control group. After 48 h of treatment, the samples were analysed by flow cytometry. Cell density was adjusted to 1 × 107 cells/mL, then macrophages incubated with FITC anti‐human CD14, PE anti‐human CD68, APC anti‐human CD209, PE anti‐human CD163, Cy5 anti‐human CD206 (BioLegend). Finally, the cells were detected with flow cytometry (Cyto‐Flex Beckman).
Fitc anti human cd14
Manufactured by BioLegend
Sourced in United States
FITC anti-human CD14 is a fluorescently labeled antibody that binds to the CD14 surface antigen, which is expressed on monocytes and macrophages. It can be used for the identification and enumeration of CD14-positive cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti human cd163, by BioLegend (2 mentions)
Apc anti human cd206, by BioLegend (2 mentions)
Apc anti human cd86, by BioLegend (2 mentions)
Recombinant human gm csf, by Miltenyi Biotec (2 mentions)
Facscalibur, by BD (2 mentions)
Anti human tim 3 pe, by BioLegend (2 mentions)
Anti mouse cd11b pe cy7, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
Pe anti human cd68, by BioLegend (1 mentions)
Apc anti human cd209, by BioLegend (1 mentions)
Beckman cytoflex, by Beckman Coulter (1 mentions)
Pe anti human cd80, by BioLegend (1 mentions)
Apc anti human cd11b, by BioLegend (1 mentions)
Permeabilization solution, by BD (1 mentions)
Fixation buffer, by BD (1 mentions)
Apc anti mouse cd206, by BioLegend (1 mentions)
Aria 3 cell sorter, by BD (1 mentions)
Pe anti mouse cd11b, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Pacific blue anti human cd3, by BioLegend (1 mentions)
21 protocols using fitc anti human cd14
1
Macrophage Phenotyping in FB Arecoline Stimulation
2
Macrophage Phenotype Analysis by Flow Cytometry
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The tMACs or iMACs were stimulated with LPS and IFN-γ for the indicated time. The single-cell suspensions were then prepared and incubated with an antibody or antibody cocktails for 15 min at room temperature for cell surface staining. Antibodies used in this study were PE Mouse IgG1, κ isotype (Biolegend, Cat: 400113, Clone: MOPC-21, Lot: B245984), APC Mouse IgG1, κ isotype (Biolegend, Cat: 400119, Clone: MOPC-21, Lot: B243042), FITC Mouse IgG1, κ isotype (Biolegend, Cat: 400107, Clone: MOPC-21, Lot: B199152), APC anti-human CD206 (Biolegend, Cat: 321109, Clone: 15-2, Lot: B348965), APC anti-human CD86 (Biolegend, Cat: 305411, Clone: IT2.2, Lot: B351349), PE anti-human CD80 (Biolegend, Cat:305208, Clone: 2D10, Lot: B330518), PE anti-human CD163 (Biolegend, Cat: 333606, Clone: GHI/61, Lot: B347256), FITC anti-human CD14 (Biolegend, Cat: 325604, Clone: HCD14, Lot: B268830) and APC anti-humanCD11B (Biolegend, Cat: 301309, Clone: ICRF44, Lot: B278346). These antibodies were used at a 1:100 dilution. Data were recorded on Beckman DxFLEX and analyzed with the FlowJo V10 software.
3
Multiparametric Flow Cytometric Analysis of Immune Cell Phenotypes
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For surface staining, cells were washed and incubated with FITC-anti-human CD14 (BioLegend), PE-anti-human CD197 (BioLegend), APC-anti-human CD206 (BioLegend), or PerCP-5.5-anti-mouse F4/80 (BioLegend), PE-anti-mouse CD11b (BioLegend), APC-anti-mouse CD206 (BioLegend) in PBS containing 2% FBS at 4°C for 30 min. Isotype-matched immunoglobulin served as controls. For intracellular staining, cells were fixed with fixation buffer (BD Biosciences) for 30 min at 4°C. The fixed cells were permeabilized with the permeabilization solution (BD Biosciences) at room temperature for 30 min. Cells were incubated overnight with Alexa Fluor 488-anti-mouse iNOS (eBioscience) at 4°C. Then, labeled cells were sorted by using the Aria III cell sorter (BD Biosciences) or analyzed by using BD FACSCanto II (BD Biosciences) with FACSDiva software.
4
Multiparametric Phenotyping of Stimulated PBMCs
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Stimulated PBMCs were recovered from the culture plates and resuspended in 100 μL PBS. Cell viability was assessed by staining with Viobility™ Fixable Dyes (Miltenyi Biotec, Germany). Cells were washed, fixed, permeabilized, and then stained with an antibody cocktail containing Pacific Blue™ anti-human CD3 (Biolegend, clone: HIT3a), PE/Cyanine7 anti-human CD4 (Biolegend, clone:A161A1) and PerCP/Cyanine5.5 anti-human CD8 (Biolegend, clone: SK1) for T cell identification; APC anti-human CD69 (Biolegend, clone: FN50) and PE anti-human IFN-γ (Biolegend, clone:4S.B3) for the activation analysis; and FITC anti-human CD14 (Biolegend, clone:HCD14) and FITC anti-human CD20 (Biolegend, clone:2H7) for the exclusion of non-specific signals and B cells. Fifty thousand events were analyzed by a BD LSR-II flow cytometer. (BD Biosciences, San Jose, CA) The gates applied for the quantification of the stimulated T cells are illustrated in Figure S1
5
Optimized Antibody Panel for Immunoblotting and Flow Cytometry
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The following antibodies were used for immunoblotting: Cell Signaling Technology, anti-p38 (Cat#: 8690), anti-p-p38 (Cat#: 4511), anti-p65 (Cat#: 8242), anti-p-p65 (Cat#: 3033), anti-JNK (Cat#: 9252), anti-p-JNK (Cat#: 4668), anti-Erk (Cat#: 4695), anti-p-Erk (Cat#: 4370), anti-PKM2 (Cat#: 4053), anti-STAT3 (Cat#: 12640); Abcam, anti-Pyk2 (Cat#: 228477); Santa Cruz Biotechnology, anti-p-Pyk2 (Cat#: sc-81512); Beyotime Institute of Biotechnology, anti-β-actin (Cat#: AA128), HRP labeled Goat Anti-Rabbit IgG (Cat#: A0208), HRP-labeled Goat Anti-Mouse IgG (Cat#: A0216). The following antibodies purchased from Biolegend were used for flow cytometry: FITC anti-mouse B220 (Cat#: 103206), BV421 anti-mouse CD11c (Cat#: 117330), FITC anti-mouse F4/80 (Cat#: 123108), APC anti-mouse CD86 (Cat#: 105012), PE anti-mouse CD40 (Cat#: 124610), PE anti-mouse GL7 (Cat#: 144607), APC anti-mouse CD95 (Cat#: 152604), APC anti-mouse CXCR5 (Cat#: 145506), PE anti-mouse PD-1 (Cat#: 135206), APC/Cy7 anti-human CD19 (Cat#: 302218), FITC anti-human CD14 (Cat#: 367116), BV421 anti-human CD11c (Cat#: 301628), APC anti-human CD86 (Cat#: 374208), PE anti-human CD40 (Cat#: 334308). All the antibodies for flow cytometry were used at a 1:100 dilution.
6
Differentiation of MUTZ-3 Cells into Immature Dendritic Cells
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The MUTZ-3 cell line derived from a human acute myelomonocytic leukaemia (DSMZ, Braunschweig, Germany) was kept in minimal essential alpha medium (MEM α, Gibco) supplemented with 20% of heat-inactivated fetal bovine serum (FBS) and 50 U/ml of recombinant human GM-CSF (Miltenyi Biotec). To differentiate MUTZ-3 cells into iDCs, 1–2 × 105 cells/ml were incubated for 7 days in the presence of 300 U/ml human recombinant GM-CSF and IL-4 (Miltenyi Biotec)22 (link). LSRII flow cytometry was applied to analyse expression of surface markers (Fig. S1B ), using FITC-anti-human CD14, PE-anti-human CD1a, PE/Cy7-anti-human DC-SIGN and APC-anti-human CD83 (all BioLegend, London, UK). Differentiated iDCs were kept in MEM α medium supplemented with 20% of heat-inactivated FBS during Aspergillus conidia interaction studies.
7
Multiparametric Flow Cytometry Panel
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Alexa Fluor® 700 anti-human CD3, (clone: HIT3a), FITC-anti human TCRαβ (clone: IP26), FITC-anti-human TCRγδ (clone: B1), APC/Cy7 Anti-Human CD127 (I-7Ra), (clone: A019D5), PE anti-human CD161, (clone: HP-3G10), Brilliant Violet 421TM anti-human CD117 (c-kit), (clone 104D2), PE/Cy7 anti-human CD294 (CRTH2), (clone: BM16), APC anti-human CD336 (NKp44), (clone: 325110), Alexa Fluor® 488 anti-human CD19, (clone: HIB19), FITC anti-human CD94, (clone:DX22), FITC anti-human CD1a, (clone: HI149), FITC anti-human CD11c, (clone: 3.9), FITC anti-human CD123, (clone: 6H6), anti-human CD303 (BDCA-2), (clone:201A), FITC anti-human CD14, (clone: 63D3), FITC anti-human FcεRIα, (clone: NP4D6), FITC anti-human CD34,(clone: 561), APC/Cy7 anti-human IFN-γ, (clone: 4S.B3) all from BioLegend. Anti-Human CD363 (S1PR1) eFluor® 660, (clone: SW4GYPP, ThermoFisher), Mouse IgG1 K Isotype Control eFluor® 660, (clone: P3.6.2.8.1, ThermoFisher). Anti-mouse CD3) APC, (clone: 17A2), anti-mouse NK1.1 Alexa Fluor® 647, (clone: PK136), anti-mouse B220 APC/Cy7 or FITC, (clone:RA3-6B2), anti-mouse CD45-Percp Cy5.5, (clone:30-F11), anti-mouse CD90.2 PE Cy7, (clone:30-H12), anti-mouse CD11b APC, (clone:M1/70), anti-mouse GATA3 PE (Clone: 16E10A23), anti-mouse Rorγt (Clone: 2F7-2).
8
Phenotyping of M1 and M2 Macrophages
9
Quantifying Immune Cell Surface Markers
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Cell surface staining of blood mononuclear cells and isolated monocytes was carried out by incubation with FITC antihuman CD14 and APC anti human CD14 (BioLegend, San Diego, CA, US). Expression of protein levels of IRAK-M was determined by flow cytometry intracellular staining. THP-1 cells and PBMCs were fixed and permeabilized (Transcription Factor Staining Buffer Set, Thermo Fisher Scientific, Waltham, MA, US). Then, staining was carried out with anti-human IRAK-M rabbit polyclonal antibody (ab-8116, Abcam) and anti-rabbit APC conjugated secondary antibody (BioLegend, San Diego, CA, US). The proper isotype control was used as a control. The flow cytometry events were acquired in a FACS Calibur (BD Biosciences, San Jose, CA) and analyzed with the use of Summit v4.3 Software.
10
Immune Cell Phenotyping and Cytokine Profiling
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PBMCs were incubated with Human TruStain FcX (BioLegend, USA) and stained with the following antibodies: FITC anti-human CD14 and Alexa Fluor 647 anti-human CD16 (BioLegend, USA). The stained cells were assessed using the FACSCelesta (BD Biosciences, USA). Data were analyzed using FlowJo software (V10.6.2., Tree Star). Nine plasma proteins, including IL-6, IL-8, CSF-1, CCL3, TNF, MCP-1, MCP-2, MCP-3, and MCP-4, were targeted and quantified by Olink multiplex proximity extension assay following the manufacturer’s instructions. The protein abundance levels were reported as normalized protein expression values on a log2 scale.
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