CD23+ B cells were purified from single cell suspensions of splenocytes with biotinylated anti-CD23 (BD Bioscience; #553137) and streptavidin microbeads (Miltenyi Biotec; #130-048-101) according to manufacturer’s instructions. Cells were cultured for 2–3 d in RPMI 1640 medium (Corning) supplemented with non-essential amino acids (Corning), 2 mM l -Glutamine (Corning), 25 mM HEPES (pH 7.2–7.6), and 50 μM β-Mercaptoethanol and stimulated with 5 μg/mL F(ab’)2 anti-mouse IgM (Jackson ImmunoResearch), 5 μg/mL purified anti-mouse CD40 (BioXcell), and 50 ng/mL IL-21 (Peprotech). For thymocyte engulfment assays68 (link) thymocytes were harvested and isolated from 4–6 week-old WT mice and treated with 50 μM Dexamethasone for 4 h to induce apoptosis. Apoptotic thymocytes were then stained with 1 μM CypHer5E (GE Healthcare) for 45 min at 37 oC in serum-free Hank’s Balanced Sodium Solution (HBSS). Stained thymocytes were co-cultured with splenocytes at a 1:10 splenocyte to thymocyte ratio. Apoptotic thymocytes were removed by washing with cold PBS and splenocytes were assessed for efferocytosis by flow cytometry. MHC-II expression was compared on efferocytic (CypHer5E+) and non-efferocytic (CypHer5E−) B cells.
Biotinylated anti cd23
Manufactured by BD
Biotinylated anti-CD23 is a laboratory reagent used for the detection and analysis of CD23, a cell surface marker. It consists of an antibody molecule that has been chemically modified to include a biotin tag. This biotin tag allows the antibody to be easily detected and quantified in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Il 21, by Thermo Fisher Scientific (4 mentions)
Non essential amino acids (neaa), by Corning (4 mentions)
L glutamine, by Corning (4 mentions)
Rpmi 1640 medium, by Corning (4 mentions)
Streptavidin microbeads, by Miltenyi Biotec (4 mentions)
Anti mouse igm f ab 2, by Jackson ImmunoResearch (2 mentions)
Cypher5e, by GE Healthcare (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Corning (2 mentions)
Penicillin, by Corning (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Ultra leafpurified anti mouse cd40, by BioLegend (2 mentions)
Imiquimod, by InvivoGen (2 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
4 protocols using biotinylated anti cd23
1
Efferocytosis Assay for B Cells
2
B Cell Activation and Proliferation Assay
3
Enrichment and Activation of Mouse B Cells
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Single-cell suspensions from pooled spleens were enriched for B cells
with biotinylated anti-CD23 (BD Bioscience) and streptavidin microbeads
(Miltenyi Biotec) following the manufacturer instructions. CD23+ B
cells were cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS
(Atlanta Biologicals), 100 U/ml Penicillin (Corning), 100 mg/ml Streptomycin
(Corning), 1X Non-Essential Amino Acids (Corning), 2 mM L-Glutamine (Corning),
25 mM HEPES (pH 7.2–7.6) and 50 μM β-Mercaptoethanol, and
stimulated with 5 μg/ml F(ab’)2 anti-mouse IgM
(αIgM; Jackson ImmunoResearch Laboratories), 5 μg/ml Ultra-LEAF
purified anti-mouse CD40 (Biolegend), in presence or absence of 50 ng/ml IL-21
(Peprotech), 1 μg/ml imiquimod (Invivogen), 10 ng/ml IL-4 (Peprotech) or
20 ng/ml IFN-γ (Peprotech). For proliferation assays, CD23+ B
cells were labelled with 2.5 μM CFSE or Cell trace violet (Invitrogen)
for 1 min at 25°C prior stimulation.
with biotinylated anti-CD23 (BD Bioscience) and streptavidin microbeads
(Miltenyi Biotec) following the manufacturer instructions. CD23+ B
cells were cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS
(Atlanta Biologicals), 100 U/ml Penicillin (Corning), 100 mg/ml Streptomycin
(Corning), 1X Non-Essential Amino Acids (Corning), 2 mM L-Glutamine (Corning),
25 mM HEPES (pH 7.2–7.6) and 50 μM β-Mercaptoethanol, and
stimulated with 5 μg/ml F(ab’)2 anti-mouse IgM
(αIgM; Jackson ImmunoResearch Laboratories), 5 μg/ml Ultra-LEAF
purified anti-mouse CD40 (Biolegend), in presence or absence of 50 ng/ml IL-21
(Peprotech), 1 μg/ml imiquimod (Invivogen), 10 ng/ml IL-4 (Peprotech) or
20 ng/ml IFN-γ (Peprotech). For proliferation assays, CD23+ B
cells were labelled with 2.5 μM CFSE or Cell trace violet (Invitrogen)
for 1 min at 25°C prior stimulation.
4
Efferocytosis and MHC-II Expression in B Cells
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+ Expand
CD23 + B cells were purified from single cell suspensions of splenocytes with biotinylated anti-CD23 (BD Bioscience) and streptavidin microbeads (Miltenyi) according to manufacturer's instructions. Cells were cultured for 2-3d in RPMI 1640 medium (Corning) supplemented with non-essential amino acids (Corning), 2mM L-Glutamine (Corning), 25mM HEPES (pH 7.2-7.6), and 50µM b-Mercaptoethanol and stimulated with 5µg/mL F(ab')2 anti-mouse IgM (Jackson ImmunoResearch), 5µg/mL purified anti-mouse CD40 (BioXcell), and 50ng/mL IL21 (Peprotech). Thymocyte engulfment assays were performed as previously described 68 . In brief, thymocytes were harvested and isolated from 4-6 week-old WT mice and treated with 50µM Dexamethasone for 4hr to induce apoptosis. Apoptotic thymocytes were then stained with 1µM CypHer5E (GE Healthcare) for 45min at 37 o C in serum-free Hank's Balanced Sodium Solution (HBSS). Stained thymocytes were co-cultured with splenocytes at a 1:10 splenocyte to thymocyte ratio. Apoptotic thymocytes were removed by washing with cold PBS and splenocytes were assessed for efferocytosis by flow cytometry. MHC-II expression was compared on efferocytic (CypHer5E + ) and non-efferocytic (CypHer5E -) B cells.
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