Fresh cells (i.e., without cryopreservation) were washed and stained for 30 min in ice-cold PBS containing 0.25% FBS. The following anti-human antibodies were used - CD14 (M5E2) (301830, BioLegend), CD16 (3G8) (302048, BioLegend) HLA-DR (L243) (307654, BioLegend). To exclude lineage positive cells, a lineage cocktail (CD3− UCHT1, CD19-HIB19, CD20-2H7, CD56-5.1H11) (363601, BioLegend), CD66b- G10F5 (305118, BioLegend), NKp30-P30-15C (325210, BioLegend) was used. The fixable viability dye e-Fluor 780 (E-Bioscience; 65-0865-18) was used to exclude dead cells during the analysis. For intracellular dengue virus staining cells are fixed after surface staining with IC fixation Buffer (e-bioscience, 00-8222-49) buffer for 10-15 minutes followed by permeabilization with perm buffer (e-bioscience, 00-8333-56) and then incubated for 45-60 minutes with anti-dengue antibody (MERCK Millipore, MAB-8705-K) that were diluted in the perm wash buffer. Then washed once with 1X perm buffer and twice with 0.25% facs buffer. Flow cytometry data acquisition was performed on a BD FACSCanto II or BD LSR-II or BD LSRFortessaTM X-20 (Becton Dickinson), and the data was analyzed using FlowJo V10 software (BD).
Cd66b g10f5
Manufactured by BioLegend
CD66b (G10F5) is a cell surface marker that is expressed on the surface of human granulocytes, including neutrophils, eosinophils, and basophils. It is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family and functions in cell-cell adhesion and signaling.
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Lab products found in correlation
Cd14 m5e2, by BioLegend (4 mentions)
Cd16 3g8, by BioLegend (4 mentions)
Hla dr l243, by BioLegend (3 mentions)
Lsrfortessa, by BD (2 mentions)
Igg2b, by BD (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Cd4 okt4, by BioLegend (2 mentions)
Cd11c 3, by Thermo Fisher Scientific (2 mentions)
Cd123 6h6, by BioLegend (2 mentions)
Cd45 hi30, by BioLegend (2 mentions)
Cd56 hcd56, by BioLegend (2 mentions)
Cd20 2h7, by BioLegend (2 mentions)
Cd8b 2st8.5h7, by Beckman Coulter (2 mentions)
Facscanto 2, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
Il 10 clone jes3 19f1, by BD (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Facsdiva, by BD (1 mentions)
9 protocols using cd66b g10f5
1
Comprehensive Immune Cell Immunophenotyping
2
Multicolor Flow Cytometry Phenotyping
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Fluorochrome-labeled monoclonal antibodies used for the phenotypic and intracellular staining assay included CD3 (clone UCHT1), CD4 (clone RPA-T4), CD8 (clone SK1), CCR7 (clone 3D12), CXCR4 (clone12G5), CCR5 (clone 2D7), CD8 (clone RPA-T8), IFNγ (clone B27), TNFα (clone MAb11), MIP-1ß (clone D21-1351) from Becton-Dickinson Pharmingen (San Diego, CA); CD45RA (2H4), CD4 (clone T4D11) from Beckman Coulter (Fullerton, CA); CD38 (clone HB7), CD107a (clone H4A3), and IL10 (clone JES3-19F1) from BD Biosciences (San Jose, CA); HLADR (clone TU36), aqua amine reactive dye from Invitrogen (Carlsbad, CA); CD66b (G10F5) Biolegend (San Diego, CA); and IL17 (clone eBio64CAP17), IL2 (clone MQ1-17H12), from eBioscience (San Diego, CA). Optimum antibody titers were determined empirically for each antibody based on preliminary titration experiments using serial dilutions, which included the manufacturers’ recommended amounts. Flow cytometry data were acquired on an LSRII (BD Immunocytometry Systems, San Jose, CA) equipped with 405, 488, and 643 nm lasers and utilizing FACSDIVA software (BDIS). Analysis of cytometry data was done with FlowJo software (TreeStar, Ashland, OR). Results were recorded as the percentage of CD4+ or CD8+ T-cells expressing a given surface marker or combination of markers.
3
Characterization of Monocyte Subsets
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At each WIHS visit, PBMCs were isolated from venous blood by standardized methods, frozen, and stored in liquid nitrogen in a specimen repository. Cryopreserved PBMCs stored in liquid nitrogen were warmed in 37°C water bath, then removed and immediately diluted with 1 mL of warm cRPMI and transferred and diluted again in an additional 8mL of warm cRPMI. Samples were then washed with PBS and stained with viability reagent (Ghost DyeTM Red 710, Tonbo Bioscience) according to manufacturer’s recommendation. Samples were stained with antibody cocktail containing CD14 (M5E2, Biolegend), CD16 (3G8, Biolegend), and dump channel: CD3 (OKT3, Tonbo Bioscience), CD19 (HIB19, Tonbo Bioscience), CD56 (5.1H11, Biolegend), CD66b (G10F5, Biolegend), gated based on living cells and excluding dump channel-positive cells. Intermediate monocytes were defined as CD14+CD16+ and non-classical monocytes were defined as CD14dimCD16+. Monocytes were sorted directly into TRIzol® LS Reagent (Life Technologies) and frozen at -80°C. FCS files were exported from FACS Diva (BD Bioscience) and processed using FlowJo v10.2 (FlowJo, LLC). The quantification of intermediate and non-classical monocytes are estimated from the FACS data from all PBMC samples.
4
Phenotyping Immune Cells by Flow Cytometry
5
Multiparametric Flow Cytometry of Immune Cells
6
Quantifying Immune Cells in BALF
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BALF was analyzed for neutrophil and alveolar macrophage counts. Cell pellets were isolated by centrifugation as described above and incubated for 30 min at 4°C with antibody cocktail containing fluorescence-conjugated antibodies of 0.5 µg/ml of CD45 (clone 30-F11; Biolegend), 0.5 µg/ml of CD11b (M1/70; BD Biosciences, San Jose, CA) and 0.5 µg/ml CD66b (G10F5; Biolegend) for neutrophils and CD45, 0.5 μg/mL of CD11c (N418; Biolegend), and 0.5 μg/mL of F4/80 (BM8; Biolegend) to identify alveolar macro phages.
7
Immune Cell Profiling by Flow Cytometry
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Flow cytometry was used to determine the phenotype of the human MDSCs and T cells using various combinations of the following fluorochrome-conjugated mAbs: anti-human HLA-DR(TU36), CD11b (ICRF44), and CD33 (WM53) were from BD Biosciences; CD14 (M5E2), CD4 (RPA-T4), IFN-γ (4S.B3), CD25 (BC96), and CD66b (G10F5) were from BioLegend; FOXP3 (236A/E7), IL-17A (eBio64DEC17), and IL-4 (8D4-8) were from ThermoFisher Scientific. Isotype controls used included mouse IgG1 (RMG1-1, MOPC-21), IgG2b (MPC-11), and IgM (MM-30) from BioLegend, mouse IgG1 (MOPC-21), IgG2a (R19-15), and IgG2b (27 (link)–35 (link)) from BD Biosciences. For intracellular staining, the cells were first stained for surface antigens, fixed, permeabilised with intracellular fixation and permeabilisation buffer (eBiosciences, San Diego, CA, USA), followed by staining with fluorochrome-conjugated mAb against the relevant intracellular proteins. All samples were collected on a fluorescence-activated cell sorter (FACS; LSRFortessa, Becton Dickinson, Franklin Lakes, NJ, USA) and analysed using the FlowJo software. Isotype controls and gating strategy for the flow cytometric analyses are presented in the Supplementary Materials (Figures S1, S2 ).
8
Multiparametric Phenotyping of Decidual Leukocytes
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1-2 X 106 fresh decidual leukocytes were washed with PBS and stained using the following cocktail of antibodies: CD45 (HI30, Biolegend), CD66b (G10F5, BioLegend), CD20 (2H7, BioLegend), CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD56 (HCD56, Biolegend), CD16 (3G8, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend), for 20 min in dark at 4C. Samples were washed twice in FACS buffer and resuspended in 400 uL. All samples were acquired with the Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA), immediately after addition of SYTOX Red Dead Cell Stain (1:1000). Data were analyzed using FlowJO (Ashland OR).
9
Comprehensive Immune Profiling of Decidual Leukocytes
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1–2 × 106 fresh decidual leukocytes were washed with PBS and stained using the following cocktail of antibodies: CD45 (HI30, Biolegend), CD66b (G10F5, BioLegend), CD20 (2H7, BioLegend), CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD56 (HCD56, Biolegend), CD16 (3G8, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend), for 20 minutes in dark at 4C. Samples were washed twice in FACS buffer and resuspended in 400 uL. All samples were acquired with the Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA), immediately after addition of SYTOX Red Dead Cell Stain (1:1000). Data were analyzed using FlowJO (Ashland OR).
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