Human PBMC purified B cells were isolated by Ficoll-Paque, followed by anti-CD19 magnetic beads (Miltenyi Biotec). Splenic naive B cells were purified using a B cell isolation kit by negative selection (Miltenyi Biotec). Splenic Pre-GC and GC B cells were first enriched by streptavidin magnetic beads conjugated with a mixture of biotinylated anti-CD11b, anti-CD11c, anti-IgD, anti-CD138, anti-CD3, and anti-Ter119 (Miltenyi Biotec), followed by cell sorting using a BD FACSAria III Cell Sorter. Splenic Pre-GC cells defined as B220+ IgD− CD38+ GL7+ cells were isolated from PyNL-infected mice on day 9 after infection. Splenic mature GC B cells defined as B220+ IgD− CD38− GL7+ cells were purified from mice recovered from PyNL infection after day 28 infection.
Anti cd138
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD138 is a monoclonal antibody that binds to the CD138 (Syndecan-1) antigen expressed on plasma cells. It is commonly used for the identification and isolation of plasma cells in flow cytometry and cell sorting applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3 cell sorter, by BD (1 mentions)
B cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti cd11b, by Miltenyi Biotec (1 mentions)
Anti cd19 magnetic beads, by Miltenyi Biotec (1 mentions)
Ficoll paque, by Miltenyi Biotec (1 mentions)
Anti igd, by Miltenyi Biotec (1 mentions)
Anti cd11c, by Miltenyi Biotec (1 mentions)
Anti ter 119, by Miltenyi Biotec (1 mentions)
Anti cd3, by Miltenyi Biotec (1 mentions)
Magnetic microbead, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Rpmi 8226 mm cell line, by American Type Culture Collection (1 mentions)
Anti cd138 microbeads, by Miltenyi Biotec (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Anti cd31, by Miltenyi Biotec (1 mentions)
Antibiotic antimycotic, by Euroclone (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Cd138 apc, by BD (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti cd138
1
Isolation of Murine B Cell Subsets
2
Isolation and Co-culture of BM-derived Endothelial and Myeloma Cells
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Primary BM MGECs, MMECs, and MM cells were obtained by Ficoll gradient centrifugation of heparinized BM aspirates followed by incubation with magnetic microbeads coated with anti-CD31 or anti-CD138, respectively (Miltenyi Biotec, Bergisch Gladbach, Germany). MGECs or MMECs were cultured in Dulbecco's modified Eagle medium (DMEM) with 20% fetal bovine serum (FBS) (Sigma-Aldrich, St Louis, MN) and 1% antibiotic/antimycotic (Euroclone, Milan, Italy). Primary CD138+ MM cells were cultured in RPMI-1640 with 10% FBS and 1% antibiotic/antimycotic. RPMI-8226 MM cell line was purchased from ATCC (Manassas, VA) and cultured in RPMI-1640 medium with 10% FBS, sodium pyruvate 1 mM, high glucose, and HEPES 1 mM (all from Sigma-Aldrich).
In co-culture experiments, MMECs were cultured with RPMI-8226 cells or primary CD138+ MM cells at a 1:5 cell ratio in the presence/absence of transwell (0.4-μm pore size, Costar, Cambridge, MA). After co-culture experiments without transwell, MM cells were immunomagnetically depleted with anti-CD138 microbeads (Miltenyi Biotec); hence, experiments were carried out with purified MMECs.
In co-culture experiments, MMECs were cultured with RPMI-8226 cells or primary CD138+ MM cells at a 1:5 cell ratio in the presence/absence of transwell (0.4-μm pore size, Costar, Cambridge, MA). After co-culture experiments without transwell, MM cells were immunomagnetically depleted with anti-CD138 microbeads (Miltenyi Biotec); hence, experiments were carried out with purified MMECs.
3
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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