Red blood cell indices were measured by an automated hematology analyzer. Leukocyte populations in blood were assessed by flow cytometry as follows: neutrophils Gr-1+, T cells CD3+, B cells B220+, monocytes F4/80+ Gr-1−, eosinophils CCR3+ SSChi, basophils FcεRIα+ CD49b+. Peritoneal cells were isolated by peritoneal lavage with PBS. MCs were identified by staining peritoneal cells for FcεRIα and CD117 (c-kit). RBCs were lysed in ACK buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1mM EDTA). Cells were blocked with anti-CD16/32 for 10 min on ice, before staining with antibodies for flow cytometry for 30 min on ice. Propidium iodide (BD Biosciences, San Jose, California, USA) was used to exclude dead cells from analysis. Antibodies used were: FcεRIα (MAR-1; eBiosciences, San Diego, California, USA), CD117 (2B8; BD Biosciences), CD49b (DX5; Biolegend, San Diego, California, USA), CCR3 (TG14; Biolegend), B220 (RA3–6B2, BD Biosciences), CD3ε (145–2C11; eBiosciences), Gr-1 (RB6–8C5; eBiosciences), F4/80 (BM8; eBiosciences), CD16/32 (2.4G2, Bio X Cell, West Lebanon, New Hampshire, USA).
Fcεriα mar 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
FcεRIα (MAR-1) is a mouse monoclonal antibody that specifically binds to the high-affinity Fc receptor for IgE (FcεRI) expressed on the surface of mast cells and basophils. This antibody can be used to detect and quantify FcεRI expression on these cell types.
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Lab products found in correlation
Gr 1 rb6 8c5, by Thermo Fisher Scientific (2 mentions)
Cd117 2b8, by BD (2 mentions)
F4 80 bm8, by Thermo Fisher Scientific (2 mentions)
Cd49b dx5, by BioLegend (2 mentions)
Ccr3 tg14, by BioLegend (2 mentions)
Cd3ε 145 2c11, by Thermo Fisher Scientific (2 mentions)
Cd16 32 2.4g2, by BioXCell (2 mentions)
Propidium iodide, by BD (2 mentions)
B220 ra3 6b2, by BD (2 mentions)
Anti human igg pe, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Streptavidin pe, by BD (1 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Rorγt q31 378, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Lymphocyte separation medium, by MP Biomedicals (1 mentions)
4 protocols using fcεriα mar 1
1
Comprehensive Immune Cell Profiling
2
Multiparametric Flow Cytometry Analysis
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Cells were stained with fluorescein-conjugated mAbs to mouse CD3ε (145-2C11), CD4 (RM4-5), CD8α (53-6.7), CD11b (M1/70), CD11c (HL3), CD49b (HMα2), B220 (RA3-6B2), CD117 (2B8), isotype-matched control mAb (BD Biosciences), FcεRIα (MAR-1) (eBioscience, San Diego, CA). For CD200R3 staining, cells were stained with biotinylated mAb to CD200R3A (6C4H2), which was generated in our laboratory as described previously [13] (link), plus streptavidin-PE (BD Biosciences). For staining with recombinant fusion proteins, cells were stained with Fc fragment of human IgG (huIgFc) or CD200-huIgFc [13] (link) followed by anti-human IgG-PE (eBioscience). Fluorescence staining was analyzed with a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR).
3
Comprehensive Immune Cell Profiling
4
Isolation and Analysis of Mononuclear Cells
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Mononuclear cells were isolated from colonic lamina propria and Peyer’s patches, as previously described.25 (link) Briefly, epithelium was dissociated by using 0.5 mM EDTA and the tissues were further treated with 1.25 mg/mL of collagenase at 37 °C.25 (link) To collect mononuclear cells from mesenteric lymph nodes and spleen, the tissues were mashed mechanically and filtered through 70 μm mesh. Lymphocyte separation medium (MP Biomedicals, Santa Ana, CA, USA) was used to isolate peripheral blood mononuclear cells (PBMCs).
For flow cytometric analysis, cells were incubated with 5 µg/mL of an anti-CD16/32 antibody (Fc block, BD Pharmingen, San Diego, CA) for 5 min and stained for 30 min at 4 °C with fluorescence-labeled antibodies (Abs) specific for c-kit (2B8), CD25 (3C7), CD45 (30F-11), CD63 (5A9),11 (link) RORγt (Q31–378) (BD Pharmingen), and FcεRIα (MAR-1) and Foxp3 (FJK-16s) (eBioscience, San Diego, CA). CD4 (RM4–5), CD11b (M1/70), CD11c (N418), CD39 (Duha59), CD45 (30F11), CD49b (DX5), CD73 (TY/11.8), CXCR5 (L138D7), PD-1 (RMP1–30), GATA3 (16E10A23), and Tim-4 (F31-5G3) were purchased from BioLegend (San Diego, CA). Cells were analyzed by using FACSCalibur, FACSCanto II, and FACSAria III flow cytometry systems (Becton Dickinson, San Jose, CA, USA). The full gating strategies for MCs is shown in Supplementary Fig.10 .
For flow cytometric analysis, cells were incubated with 5 µg/mL of an anti-CD16/32 antibody (Fc block, BD Pharmingen, San Diego, CA) for 5 min and stained for 30 min at 4 °C with fluorescence-labeled antibodies (Abs) specific for c-kit (2B8), CD25 (3C7), CD45 (30F-11), CD63 (5A9),11 (link) RORγt (Q31–378) (BD Pharmingen), and FcεRIα (MAR-1) and Foxp3 (FJK-16s) (eBioscience, San Diego, CA). CD4 (RM4–5), CD11b (M1/70), CD11c (N418), CD39 (Duha59), CD45 (30F11), CD49b (DX5), CD73 (TY/11.8), CXCR5 (L138D7), PD-1 (RMP1–30), GATA3 (16E10A23), and Tim-4 (F31-5G3) were purchased from BioLegend (San Diego, CA). Cells were analyzed by using FACSCalibur, FACSCanto II, and FACSAria III flow cytometry systems (Becton Dickinson, San Jose, CA, USA). The full gating strategies for MCs is shown in Supplementary Fig.
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