To isolate DCs, spleens were harvested, incubated 30 min at 37 °C in RPMI, 2% FBS, 20 mM HEPES, 400 U/ml type IV collagenase (Worthington Biochemical Corporation) and disrupted to generate single cell suspensions. Red blood cells were lysed with ACK buffer (Lonza), and the resulting cell suspensions were filtered through a 70 μm mesh into PBS supplemented with 0.5% BSA and 2 mM EDTA (PBE). DCs were obtained by magnetic cell separation (MACS) using anti-CD11c beads (Miltenyi Biotec), as per manufacturer’s instructions. To isolate CD4+ T cells and B cells, spleens were processed as above, except for collagenase digestion, which was not performed. CD4+ T cells were isolated using CD4+ T cell isolation kit (Miltenyi Biotec) while B cells were obtained by negative selection using anti-CD43 beads (Miltenyi Biotec), as per manufacturer’s instructions. To isolate Igλ+ B cells form B1-8hi mice, B cells were stained with anti-Igκ PE antibody and subsequently purified by negative selection using a combination of anti-CD43 and anti-PE magnetic beads (Miltenyi Biotec).
Anti cd43
Manufactured by Miltenyi Biotec
Anti-CD43 is a monoclonal antibody that recognizes the CD43 surface antigen. CD43 is a sialoglycoprotein expressed on the surface of various hematopoietic cells, including T cells, B cells, monocytes, and granulocytes. The primary function of the Anti-CD43 antibody is to enable the detection and identification of CD43-positive cells in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11c beads, by Miltenyi Biotec (1 mentions)
Anti cd43 beads, by Miltenyi Biotec (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Ack buffer, by Lonza (1 mentions)
Anti pe magnetic beads, by Miltenyi Biotec (1 mentions)
Cd138 apc, by BD (1 mentions)
Cd40l, by BD (1 mentions)
Cd40l, by Miltenyi Biotec (1 mentions)
Anti cd138, by Miltenyi Biotec (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (1 mentions)
Dead cell removal kit, by Miltenyi Biotec (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Sodium pyruvate, by Corning (1 mentions)
Cd40l, by R&D Systems (1 mentions)
2 protocols using anti cd43
1
Isolation of Immune Cell Populations
2
Isolation and Differentiation of Murine Naive B Cells
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Whole spleens were mechanically disassociated through a 40 m filter followed by red blood cell lysis with ammonium-chloride-potassium lysing buffer. Naïve mature B cells were isolated from spleen cells by immunomagnetic negative selection with anti-CD43 (Miltenyi Biotec). B cells were cultured in RPMI-1640 medium (Corning) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Corning), 1% MEM non-essential amino acids (Gibco), 1% sodiumpyruvate (Corning), 1% L-glutamine (Gibco), and -mercaptoethanol (50 M). B cells were stimulated with 1 ug/ml CD40L (BD Pharmingen) and 25 ng/ml IL-4 (R&D Systems) or with 0.1 ug/ml CD40L, 10 ng/ml IL-4, and 5 ng/ml IL-5 (R&D Systems) to monitor B cell processes during B cell differentiation or to monitor differentiation with increased plasma cell (PC) frequency, respectively (Shi et al., 2015) . For isolation of in vitro generated PCs used for Seahorse Extracellular Flux Analyzer assays, cultures stimulated for 5 days with CD40L, IL-4, and IL-5 were enriched for live cells using a dead cell removal kit (Miltenyi Biotec) followed by CD138-APC (BD Pharmingen) staining. CD138 + PCs were enriched by positive immunomagnetic enrichment of APC (Miltenyi Biotec). For Caspase 3/7, Mitrotracker Green, and TMRE staining experiments, in vitro generated PCs were isolated by immunomagnetic positive selection with anti-CD138 (Miltenyi Biotec).
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