Whole naïve or granulomatous livers were chopped into fine pieces with a razor blade and digested in 100 units/ml collagenase (Sigma) for 1 hr at 37oC with rocking. The tissue was then ground into a single-cell suspension through a 100-µm nylon mesh. Hepatocytes were pelleted out with a 50 g spin for 5 min for cleaner density separation. Leukocytes were separated on a 40% Percoll (Sigma-Aldrich) gradient (2000 rpm for 15 min) and treated for 2 min with 1 ml ACK (ammonium chloride–potassium bicarbonate) lysis buffer to lyse erythrocytes. Leukocytes were stained for 30 minutes with antibodies for CD16/32 (BDBiosciences), CD45 (Biolegend; San Diego, CA), CD11b (Biolegend), Siglec F (BDBiosciences), Ly6G (BDBiosciences), F4/80 (Biolegend), CD64 (Biolegend), and Ly6C (Biolegend) diluted in FACS buffer. CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ cells were sorted with at least 90% purity from amongst the stained cells using a FACS Aria (BDBiosciences).
Siglec f
Manufactured by BD
Sourced in United States, United Kingdom, Germany
Siglec-F is a cell surface receptor that belongs to the sialic acid-binding immunoglobulin-like lectin (Siglec) family. It is primarily expressed on eosinophils and plays a role in the regulation of eosinophil function and homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BD (25 mentions)
Cd11c, by BD (18 mentions)
Cd11b, by BioLegend (16 mentions)
Cd11c, by BioLegend (16 mentions)
F4 80, by BD (13 mentions)
Cd11c, by Thermo Fisher Scientific (12 mentions)
Cd11b, by Thermo Fisher Scientific (11 mentions)
F4 80, by BioLegend (8 mentions)
Cd103, by BD (7 mentions)
Ifn γ, by Thermo Fisher Scientific (7 mentions)
F4 80, by Thermo Fisher Scientific (7 mentions)
Fc block, by BD (5 mentions)
Cd206, by BioLegend (5 mentions)
Ly6g 1a8, by BioLegend (5 mentions)
Mhcii, by BioLegend (5 mentions)
Collagenase, by Merck Group (4 mentions)
Golgi stop reagent, by BD (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Il 17a, by BD (4 mentions)
Software, by FlowJo (4 mentions)
101 protocols using siglec f
1
Liver Macrophage Isolation Protocol
2
Enriching Intestinal Tuft Cells
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To enrich tuft cell populations, intestinal single-cell suspensions were prepared as described in the published paper17 (link). In brief, the small intestine was opened and rinsed with PBS to remove luminal contents. The small intestine was then incubated in 5 ml PBS containing 2.5 mM EDTA, 0.75 mM DTT, and 10 μg /ml DNaseI (Sigma) at 37 °C for 20 min with rocking. After incubation, tissues were shaken vigorously for 30 s and then incubated in 5 ml HBSS (Ca2+/Mg2+ free) containing 1.0 U/ ml Dispase (Gibco) and 10 μg/ml DNaseI at 37 °C for 10 min with rocking. Cells were filtered through a 70 μm filter to get intestinal single-cell suspensions. Intestinal single cells were stained with the EpCAM (Invitrogen, Cat#118202, 1:200 dilution) and SiglecF (BD, Cat#552126, 1:50 dilution) antibodies. EpCAM+ SiglecF+ cell populations were sorted as tuft cell-enriched populations using a BD High-Speed Cell Sorter.
3
Multiparameter Flow Cytometry Analysis
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The collected bone marrow cells, splenocytes and lung cells were suspended with staining buffer (PBS containing 3% FBS) and stained with fluorescent-conjugated antibodies specific for CCR3 (Biolegend, San Diego, CA, USA), SiglecF (BD Biosciences, Franklin Lakes, NJ, USA), Ly6G (Biolegend), CD11b (BD Biosciences), CD11c (BD Biosciences), Ly6C (BD Biosciences), CD3e (TONBO Biosciences, San Diego, CA, USA), CD4 (Biolegend), CD8 (TONBO Biosciences), CD14 (Biolegend), CD16/32 (BD Biosciences), CD19 (TONBO Biosciences), CD25 (TONBO Biosciences), CD44 (TONBO Biosciences), CD45.1 (Biolegend), CD45.2 (Biolegend), B220 (TONBO Biosciences), Gr-1 (Biolegend), Sca-1 (BD Biosciences), Il-5Rα (BD Biosciences), CD34 (BD Biosciences), and c-kit (Biolegend). The cells were sorted with a FACSAria II (BD Biosciences) and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
4
Isolation and Analysis of Pulmonary Immune Cells
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Lung tissue was incubated with stirring at 37°C for 30 min in Hank’s balanced salt solution (HBSS) with 1.3 mM EDTA (Invitrogen), then minced and followed by treatment at 37°C for 45 min. with collagenase (1 mg / ml; Sigma) in RPMI with 5% fetal calf serum (FCS) and with 100 μg / ml of DNase for 10 min. Cells were lysed with ACK (Lonza, Walkersville, MD) to remove erythrocytes. Cells were blocked with Fc Block (BD Biosciences, San Jose, CA), directly stained with fluorochrome-conjugated Abs against Ly6G (1A8), MHCII, CD11c, Siglec-F, F4/80, CD19, B220 (BD Biosciences), CD21, CD23, CD24, CD5, Cd1, CD138 (Biolegend), RELMα (Biorbyt), and analyzed by flow cytometry. Neutrophils were identified as CD11b+Ly6G+, Eosinophils as F4/80+CD11c-Siglec-F+, Alveolar macrophages as F4/80+CD11cvarSiglec-F+, nonalveolar macrophages as F4/80+CD11cvarSiglec-F-. In some experiments, B cells were sort purified (> 98%) for adoptive transfer. For B lymphocyte transfers, B cells purified from lung tissue and MLN of donor mice were transferred i.t. into recipient mice. B cells purified from spleens of donor mice were transferred intra-peritoneally (i.p.) into recipient mice. A total of three cell transfers were performed (day −3, 0, and +1) during N. brasiliensis inoculation.
5
Isolation and Analysis of Pulmonary Immune Cells
6
Multi-color Flow Cytometry for Immune Cell Profiling
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Cell staining was performed using antibodies against B220 (clone: RA3-6B2; used at 1:500, Biolegend, 103227), CD3e (clone:145-2C11; 1:500, Biolegend, 100304), CD4 (clone: GK1.5; 1:500, Biolegend, 100423), CD5 (clone:53-7.3; 1:200, Biolegend, 100604), CD8a (clone:53-6.7; 1:500, Biolegend, 100704), CD11b (clone: M1/70; 1:200, Biolegend, 101204), CD11c (clone: N418; 1:200, Biolegend, 117304), CD45.2 (clone:104; 1:500, eBioscience, 11-0454-85), CD49b (clone: DX5; 1:200, Biolegend, 108904), F4/80 (clone: BM8; 1:200, Biolegend, 123106), FceR1 (clone: Mar1; 1:200, Biolegend, 134318), Gr-1 (clone: RB6-8C5; 1:500, BD Biosciences, 553124), NK1.1 (clone: PK136; 1:300, Biolegend, 108704), Siglec-F (clone: E50-2440; 1:400, BD Biosciences, 552126), Thy1.2 (clone:30-H12; 1:500, BD Biosciences, 105324), Podoplanin (clone:8.1.1; 1:100, Biolegend, 127410), and Ter119 (1:200, eBioscience, 13-5921-82). Flow cytometric analysis and cell sorting were performed using FACSCalibur and FACSAria III systems (BD Biosciences), and data were analyzed using FlowJo software (BD Biosciences). Doublet cells were excluded by FL-2A/FL-2H plots, then FSC/SSC plots were used to narrow down eosinophil or T cell populations followed by further gating with Siglec-F/Gr-1 or Siglec-F/CD11c (for eosinophils), or CD4/ST-2 (for TH2 cells) plots (Supplementary Figure 3
7
Isolation and Characterization of Adipose-Derived Immune Cells
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Retroperitoneal adipose tissue was digested in 1 mg/ml Collagenase type II (Sigma, UK) for 45 minutes at 37C with gentle agitation and then passed through 100 M filter to generate the stromal vascular fraction (SVF). Red blood cells in MLN and SVF cell suspensions were lysed (eBioscience, UK); cells were then washed in FACS buffer (2.5% BSA; 0.5 mM EDTA, in PBS) and then incubated with Fc block (Biolegend, UK) prior to staining with the relevant antibodies/streptavidin conjugates (all BioLegend, UK unless stated otherwise). In adipose tissues, eosinophils were characterised as SiglecF+ (PE: Catalogue number 552126 BD Bioscience, UK), and constitutive IL-10 expression was identified using anti-IL-10 antibody (APC: catalogue number 505009). B regulatory cells in the MLN were identified by CD19 (AF700: Catalogue number 115527), B220 (PerCP: catalogue number 103235) and IL-10 (PE: Catalogue number 505007) expression [34] . Fixable viability stain (eFluor450:
Catalogue number 65-0863-14; eBioscience, UK) was used to select for live cells. Data was acquired using a BD LSRII flow cytometer and populations were gated using isotype and fluorescence minus one (FMO) controls using FlowJo, LLC analysis software (Tree Star/ BD) as described previously [20, 34] .
Catalogue number 65-0863-14; eBioscience, UK) was used to select for live cells. Data was acquired using a BD LSRII flow cytometer and populations were gated using isotype and fluorescence minus one (FMO) controls using FlowJo, LLC analysis software (Tree Star/ BD) as described previously [20, 34] .
8
Comprehensive Immune Cell Profiling in Lungs
9
Multiparametric Flow Cytometry of Immune Cells
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Whole-blood samples (100 μL) were incubated with Fc block (1:500; BioLegend) for 10 min and then incubated with the following antibodies for 15 min: CD45 (1:200; BioLegend); CD11b (1:200; BioLegend); Ly6C (1:100; BioLegend); Ly6G (1:200; BioLegend); CD115 (1:100; BioLegend); NK1.1 (1:100; BD BioSciences); B220 (1:200; BioLegend); and Siglec F (1:100; BD BioSciences). Next, samples were lysed with 1X Red Blood Cell Lysis Buffer (BioLegend) and washed with FACS buffer. Cell populations were sorted with a BD FACSCanto II Cell Analyzer (BD Biosciences). Briefly, 80,000 CD45+ events were collected. 7-aminoactinomycin D (7-AAD) was used to separate live and dead cells. CD45+ cells were then gated on CD11b+ events, then gated on Ly6C, Ly6G, and CD115. To isolate monocyte and neutrophil populations, CD3, B220, Siglec F, and NK1.1 were used to remove T cells, B cells, eosinophils, and natural killer cells, respectively. Cell populations are presented as the number of live events in Supplementary Table S2 . The total number of events of interest was taken as a ratio to CD11b+ cells to calculate the percentage of cells of interest. A diagram of the gating strategy is provided in Figure 2A .
10
Multiparameter Flow Cytometry Analysis
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Cells isolated from the BALF and single cell suspension of lungs were stained with fluorochrome-conjugated antibodies CD45, CD11b, Ly6G, F4/80, CD11c, CD4, CD8 (Biolegend), SiglecF (BD Biosciences) or MHC-I tetramers, MHC-II tetramer (NIH Tetramer Core at Emory University). Cells were incubated with fluorochrome-conjugated antibodies for 30 minutes at room temperature prior to washing and fixing with 1% formaldehyde. For intracellular cytokine staining, splenocytes were re-stimulated with PMA and ionomycin for 5 hours and Golgi inhibitor monesin (Biolegend) was added for the last 3 hours. After re-stimulation, cells were stained with surface marker including viability dye, washed, fixed and permeabilized and stained with intracellular antibody IFN- γ (Biolegend) for 1 hour. Stained cells were analyzed on an Attune Flow Cytometer (Thermo Fisher).
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