Procedures for staining cells with antibodies for flow cytometry have been described in detail in earlier studies.15 Anti‐CD16/32 (FcBlock: 5 µg/mL) (Biolegend, San Diego, CA, USA) was used to block non‐specific antibody binding through Fc receptors. Fluorochrome‐ or biotin‐conjugated antibodies used were specific for CD11b, (M1/70), CD11c (N418), Ly6C (Al‐21), Ly6G (1A8), CD8 (53‐6.7), CD43 (1B11) and Siglec‐F (E50‐2440) (Biolegend). Propidium iodide staining (PI; 1 µg/mL) prior to flow cytometry was used to identify and gate live (PI−) cells. Flow cytometric analysis of labelled subsets was performed on a BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). FACSDiva software (Becton Dickinson) was used to acquire data. FloJo software (Tree Star, Ashland, OR, USA) was used for data analysis.
Siglec f e50 2440
Manufactured by BioLegend
Sourced in United States
Siglec‐F (E50‐2440) is a mouse monoclonal antibody that recognizes the Siglec-F protein. Siglec-F is a sialic acid-binding immunoglobulin-like lectin that is expressed on eosinophils and functions in the regulation of eosinophil-mediated inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd16 32 fc block, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Flojo software, by Tree Star (1 mentions)
Cd43 1b11, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Cd8a 53 6, by BD (1 mentions)
Cd11b m1 70, by BD (1 mentions)
Cd11c n418, by BioLegend (1 mentions)
7 aad viability staining solution, by BioLegend (1 mentions)
F4 80 bm8, by BioLegend (1 mentions)
Ly6g 1a8, by BioLegend (1 mentions)
Cd115 afs98, by BioLegend (1 mentions)
Cd206 c068c2, by BioLegend (1 mentions)
Ly6c al 21, by BD (1 mentions)
Dapi solution, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Collagenase, by Roche (1 mentions)
Golgistop, by BD (1 mentions)
4 protocols using siglec f e50 2440
1
Antibody Staining Protocol for Flow Cytometry
2
Immune Cell Phenotyping by Flow Cytometry
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Cells were incubated with CD16/CD32 Fc blocking antibody (2.4G2, 1:100; BD Biosciences, San Jose, CA, USA) for 10 min, then stained with fluorochrome-conjugated antibodies for 30 min on ice. CD11c (N418, 1:200), Ly6G (1A8, 1:300), CD115 (AFS98, 1:300), CD206 (C068C2, 1:400), F4/80 (BM8, 1:300), MHC II (M5/114, 1:300), Siglec-F (E50-2440, 1:300), and 7-AAD Viability Staining Solution were purchased from BioLegend (San Diego, CA, USA). CD11b (M1/70, 1:400), Ly6C (AL-21, 1:200), CD8a (53-6.7, 1:200), CD3 (17A2, 1;300), DAPI solution, and Annexin V were purchased from BD Biosciences. We performed flow cytometry with an LSRFortessa flow cytometer and sorted cells with an FACSAria III cell sorter (both from BD Biosciences). For data analysis, we used FlowJo software version 10.4 (Ashland, OR, USA).
3
Quantifying Immune Cell Infiltration in Gelatin Sponge
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Gelatin sponge implanted with 100 μL PBS or QR‐32 cells (4 × 105 cells) in mice was excised and digested for 1 h at 37°C with 2 mg/mL collagenase (Roche, Basel, Switzerland) in serum‐free RPMI. Obtained gelatin sponge‐infiltrating cells were used in other experiments. Gelatin sponge‐infiltrating cells were stained in the presence of anti‐CD16/32 (2.4G2), with some of the following antibodies: anti‐NK1.1 (PK136), CD3ε (145‐2C11), CD4 (RM4‐5), γδTCR (GL3), CD27 (LG.3A10), CCR6 (29‐2L17), CD103 (2E7), CD11b (M1/70), Ly‐6G (1A8), F4/80 (BM8), CD25 (7D4) and/or Siglec‐F (E50‐2440) antibodies purchased from Biolegend (San Diego, CA, USA) or BD Pharmingen (San Jose, CA, USA). Flow cytometry analysis was performed by FACSAria cell sorter, or FACS Canto (BD Bioscience, San Jose, CA, USA) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). To analyze the intracellular level of IL‐17, gelatin sponge‐infiltrating cells were incubated in the presence of GolgiStop (BD Bioscience) for 4 h without in vitro stimulation. After surface marker staining, cells were fixed and permeabilized with the BD Cytofix/Cytoperm Kit (BD Bioscience) according to the manufacturer's instructions. Then, cells were stained with anti‐IL‐17 (TC11‐18H10.1) antibody purchased from Biolagend and analyzed by flow cytometry.
4
Murine Immune Cell Profiling
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MA (MW 1,100–1,300 Da) was purchased from Sigma-Aldrich (St. Louis, MO) and reconstituted in an organic solution that comprised of chloroform and methanol at a ratio of 3 to 1 and stored as aliquots at −20°C. Monoclonal antibodies against mouse CD11b (M1/70), CD11c (N418), NK1.1 (PK136), CD19 (6D5), Ly6G (1A8), CD25 (PC61), CD44 (1M7), CD69 (H1.2F3), CD103 (2E7), F4/80 (BM8), TCRβ (H57-597), Siglec F (E50-2440) and human TCR Vβ5.1 (LC4) with different fluorochrome conjugates were purchased either from BioLegend or eBioscience (San Diego, CA).
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