For B cell enrichment, splenocytes were incubated with antibody cocktail containing biotin-labeled αCD90, αCD11c, and αCD11b (eBioscience). Anti-biotin microbeads (Miltenyi Biotec) were used to deplete CD90+ T-cells, CD11c+ DCs, and CD11b+ macrophages using MACS separation. Purified B-cells were left unstimulated or were stimulated with α-IgM (Jackson ImmunoResearch) at 10μg/ml plus IL-4 (Peprotech) at 20ng/ml, α-CD40 (BD Biosciences) at 10μg/ml plus IL-4 at 20ng/ml, LPS (Sigma Aldrich; 055:B5) at 5μg/ml and CpG (Invioogen; ODN 1826) at 5μg/ml. Kyn/Trp ratio was determined with High-Performance Liquid Chromatography (HPLC) analysis of culture medium as previously described (28 (link)). Cytokine protein concentrations in the culture medium were measured using Ready-Set-Go! ELISA kits (eBioscience). Cell proliferation was determined by FACS analysis after labeling cells with 0.5μM 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen). Cell survival was quantified on flow cytometer by examining annexin V staining. To test the surface expression profiles of activation markers, B cells were stained with α-CD86 (BD Pharmingen), α-MHC class II (eBioscience), α-IgM (Biolegend), α-CD44 (BD Pharmingen), α-IgM (Biolegend), α-CD40 (Biolegend). Events were collected on LSR II flow cytometer and all results were analyzed with FlowJo software (TreeStar).
α cd11b
Manufactured by Thermo Fisher Scientific
α-CD11b is a lab equipment product. It is a protein that binds to the CD11b antigen. CD11b is a component of the integrin family of cell surface receptors.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
α cd40, by BioLegend (1 mentions)
αcd11c, by Thermo Fisher Scientific (1 mentions)
α cd40, by BD (1 mentions)
α igm, by BioLegend (1 mentions)
α cd44, by BD (1 mentions)
Interleukin 4 (il 4), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Anti biotin microbead, by Miltenyi Biotec (1 mentions)
Cytofix cytoperm protocol, by BD (1 mentions)
αifnγ xmg1.2, by Thermo Fisher Scientific (1 mentions)
Live dead fixable yellow, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
αcd11c, by BioLegend (1 mentions)
Mopc 21, by BioLegend (1 mentions)
αcd29, by R&D Systems (1 mentions)
Clone mab11, by BioLegend (1 mentions)
3 protocols using α cd11b
1
B Cell Enrichment and Activation Assay
2
Multiparameter Immune Cell Profiling
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Single-cell suspensions (1 × 106 cells) was incubated at 4° C for 15 min with α-CD16/32 (clone 2.4G2, from Bioexpress) to reduce non-specific binding. Cells were labeled with various combinations of directly conjugated monoclonal antibodies and incubated at 4°C for 25 min. The following monoclonal antibodies were used: α-NK1.1 (PK136), α-CD3 (145-2C11), α-TCRβ (H57-597), α-CD8a (53–6.7), α-CD49b (DX5), α-CD27 (LG.7F9), α-CD11b (M1/70), α-Ly49D (4E5), α-Ly49G2 (4D11), α-Ly49H (3D10), α-NKG2A-B6 (16a11), α-NKG2D (CX5), α-IL-10 (JES5-16E3), α-IFNγ (XMG1.2), and α-CD107a (1D4B) from eBioscience, α-CD19 (1D3), α-Ly49C/I (5E6), α-CD4 (RM4-5), and α-CD49a (Ha31/8) from BD Biosciences, Fixable Yellow Live/Dead from Invitrogen. For intracellular IL-10 and IFNγ measurements in vivo, leukocytes were harvested and incubated in RP-10 medium (RPMI-1640, 10% FBS, 1X penicillin/streptomycin, 1% L-Glutamine, 10 mM HEPES, 50 μM β-mercaptoethanol) containing 5 μg/ml brefeldin A for 4 h, followed by staining for intracellular IL-10 or IFNγ using BD Cytofix/Cytoperm protocol. The stained cells were acquired using LSRFortessa or FACSCelesta (BD Biosciences) and analyzed using Kaluza software v3 (Beckman Coulter) or FlowJo (Tree Star).
3
Blocking of Fc Receptors in Immunoassays
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The chimeric α-ganglioside GM2 monoclonal antibody (KM966) was a kind gift from Kyowa Hakko Kirin Co., Ltd. The chimeric α-CD20 (rituximab) and the humanized α-HER2 (trastuzumab) were purchased from Roche Pharmaceuticals. The TCR-like antibodies were generated in laboratory of Antonio Bertoletti. α-CD64 (clone 10.1, BioLegend), α-CD32 (clone AT10, Abcam), α-CD32a (clone IV.3, StemCell Technologies), α-CD32b (polyclonal, Abcam), α-CD16 (clone DJ130c, AbD Serotec), α-CD18 (Clone TS1/18, Biolegend), α-CD11a (Clone TS1/22, Thermo Scientific), α-CD11b (Clone CBRM1/5, eBioscience), α-CD11c (Clone 3.9, Biolegend), α-CD29 (Clone P5D2, R&D systems), α-TNFα (Clone Mab11, Biolegend), α-TNFR (Clone mab625, R&D systems) and an isotype-matched control antibody (MOPC21, BioLegend) were used for blocking experiments.
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