Pan B cells were isolated from spleens of Sox8−/− or littermate control mice using MojoSort mouse pan B cell isolation kit (BioLegend) in accordance with the manufacturer’s instructions. For IgA+ B cell polarization, pan B cells were stimulated with 2 µg/ml F(ab′)2-goat-anti-mIgM (Thermo Fisher Scientific), 5 µg/ml anti-mCD40 (3/23; BioLegend) in complete advanced RPMI 1640 media (Thermo Fisher Scientific) containing 5% vol:vol FCS (MP Biomedicals) supplemented with 1 ng/ml recombinant human (rh) TGF-β1, 5 ng/ml recombinant mouse (rm) IL-5, 20 ng/ml rmIL-21 (all from BioLegend), and 10 nM all-trans retinoic acid (Fujifilm Wako Pure Chemical) for 6 d. For IgG1+ and IgE+ B cell polarization, pan B cells were stimulated with anti-mIgM and anti-mCD40 in complete media supplemented with 20 ng/ml rmIL-21 and 50 ng/ml rmIL-4 (BioLegend) for 6 d. For IgG2a+ B cell polarization, pan B cells were stimulated with anti-mIgM and anti-mCD40 in complete media supplemented with 20 ng/ml rmIL-21 and 50 ng/ml rmIFN-γ (BioLegend) for 6 d.
Fetal calf serum (fcs)
Manufactured by MP Biomedicals
Sourced in United States
Fetal calf serum is a cell culture supplement derived from the blood of bovine fetuses. It provides a rich source of growth factors, nutrients, and other components essential for the cultivation of a variety of cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin, by MP Biomedicals (2 mentions)
Streptomycin, by MP Biomedicals (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Mojosort mouse pan b cell isolation kit, by BioLegend (1 mentions)
Rmil 4, by BioLegend (1 mentions)
Rmil 21, by BioLegend (1 mentions)
Anti migm, by BioLegend (1 mentions)
Anti mcd40, by BioLegend (1 mentions)
Rmifn γ, by BioLegend (1 mentions)
Accumax, by Innovative Cell Technologies (1 mentions)
Cd3 pacific blue clone sp34 2, by BD (1 mentions)
Cd8 apc h7 clone sk1, by BD (1 mentions)
Tnf α fitc clone mab11, by BD (1 mentions)
Cd4 pe cy7 clone sk3, by BD (1 mentions)
Ifnγ apc clone b27, by BD (1 mentions)
17 protocols using fetal calf serum (fcs)
1
Isolation and Polarization of B Cell Subsets
2
Establishment and Culture of CCA Stem-like Cells
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A parental CCA cell line, KKU‐055, was established from poorly differentiated primary CCA tissue of a 56‐year‐old Thai man and was obtained from the Japanese Collection of Research Bioresources Cell Bank. The cell was maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% FCS (MP Biomedicals). KKU‐055 CCA stem‐like cell (KKU‐055‐CSC) was maintained in a stem cell culture medium.18 The CSCs were subcultured once a week using Accumax (Innovative Cell Technologies). All cell lines were cultured in an incubator at 37°C with 5% CO2.
3
Profiling HCV-specific T-cell Cytokine Response
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The cytokine production profile of HCV-specific T-cell lines was determined by intracellular cytokine staining (ICS) after restimulation with NS3-peptides. For T-cell epitope mapping 0.2×106 expanded cells were either restimulated with NS3-peptide-pool (1 µg/ml/peptide) or without NS3-peptide, in medium containing co-stimulatory αCD28 and αCD49d molecules (2 µg each, BD-Biosciences) and 5% FCS (MP-Biomedicals). After 2 hrs, Brefeldin A (Golgiplug 1∶1000, BD-Biosciences) was added and 16 hours later the surface markers were stained with a panel of fluorochrome labeled antibodies, containing CD3-PacificBlue (clone SP-34-2, (BD-Pharmingen), CD14-PE-TexasRed (clone RMO52, BeckmanCoulter), CD20-PE-TexasRed (clone B9E9, BeckmanCoulter), CD4-PE-Cy7 (clone SK3, BD-Pharmingen) and CD8-APC-H7 (clone SK1, BD-Pharmingen). Stained cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences), followed by staining of accumulated intracellular cytokines with IFNγ-APC (clone B27, BD Pharmingen), IL-2-PE (clone MQ1-17H12, BD Pharmingen) and TNFα-FITC (clone MAb11, BD Pharmingen). Cell staining was analyzed on a FACSAria (BD Bioscience) and DIVA software Version 6.1.1.
To evaluate antigen sensitivity, T-cell lines were restimulated with increasing concentrations of individual peptide ranging from 0.05 to 10 µg/ml before intracellular cytokine production was measured.
To evaluate antigen sensitivity, T-cell lines were restimulated with increasing concentrations of individual peptide ranging from 0.05 to 10 µg/ml before intracellular cytokine production was measured.
4
Induction of Graft-versus-Host Disease
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For preparation of donor cells, spleens and LN (cervical, axillary, brachial, inguinal, and mesenteric) were removed from DBA/2 mice and gently passed through a 40 µm nylon mesh to obtain single cell suspensions in serum-free (SF) IMDM supplemented with 2% FCS (MP Biomedical, USA) and 0.5% Ciproxine (Bayer AG, CH). Spleen cell suspensions were treated with ACK buffer to lyse erythrocytes. Single cell suspensions were pooled, counted by Trypan blue exclusion, and washed in SF IMEM (Sigma-Aldrich, USA) prior to injection. GvHD was induced by i.v. injection of 70 × 106 DBA/2 lymphocytes in a volume of 200 µl SF-IMEM.
5
Splenocyte Chemotaxis Assay
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Chemotaxis was assessed by a modified Boyden chamber migration assay using a microplate with a 3 μm pore filter (Neuro Probe, Gaithersburg, MD). After red cell lysis (BD Pharm Lyse; BD Biosciences), freshly isolated splenocytes were rested in RPMI 1640 with 10% FCS (MP Biomedicals, Santa Ana, CA), 1% sodium pyruvate, 5 × 10–5 M 2‐mercaptoethanol, 1% HEPES, 2 mmol/L l ‐glutamine (PAA LABORATORIES, Pasching, Austria), 50 U/mL penicillin, 50 μg/mL streptomycin (Corning, Manassas, VA), for at least 4 h at 37°C prior to the assay. LPS‐free fully reduced HMGB1 (HMGBiotech s.r.l., Milan, Italy), CXCL12 (PeproTech, London, UK), and glycyrrhizin were diluted in the same medium at the indicated concentration, and added to the bottom chamber of the chemotaxis plate. HMGB1 was used at the concentration of 300 nM, as described [13 (link)]. Total splenocytes (5 × 105 per well) were added in the top wells and allowed to migrate for 3 h. The migrated cells were recovered from the bottom wells, stained with propidium iodide, and B lymphocytes were counted by flow cytometry. The migration index was calculated by dividing the number of migrated cells in each well by the number of those migrated toward medium alone.
6
Expansion and Differentiation of Progenitor Cells
7
Ciprofloxacin-Resistant Proximal Tubule Cells
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The ciPTEC line was generated as previously described by Wilmer et al. (2010) . The cells were cultured in phenol red free DMEM/F12 (Gibco/Invitrogen, Breda, the Netherlands) supplemented with 10% (v/v) fetal calf serum (FCS; MP Biomedicals, Uden, the Netherlands), insulin (5 lg/ml), transferrin (5 lg/ml), selenium (5 ng/ml), hydrocortisone (36 ng/ml), epithelial growth factor (10 ng/ml), and tri-iodothyronine (40 pg/ml) at 33 °C in a 5% (v/v) CO 2 atmosphere. Propagation of cells was maintained by subculturing the cells at a dilution of 1:3-1:6 at 33 °C. For all experiments, cells were cultured at 33 °C to 40% confluency, followed by maturation for 7 days at 37 °C. Experiments were performed on the cells between passages 30 and 40.
8
Differentiating Dendritic Cells
9
Culturing Human Cell Lines
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The 116, a Cre recombinase expressing HEK293 cell line (kindly provided by Dr. Phillip Ng)23 (link), was cultured in MEM (Nacalai tesque, Japan) with 10% fetal calf serum (FCS; MP Biomedicals, Solon, OH). The human osteosarcoma cell line, MG-63 (Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University)24 (link) was cultured in MEM with 10% FCS supplemented with 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO) and 1x non-essential amino acids (Sigma-Aldrich). The human cervical carcinoma cell line, HeLa, and HEK293 cell line were cultured in DMEM (Nacalai tesque) with 10% FCS. The human iPSC line, TIG3/KOSM (formerly termed SeVdp (KOSM) #7)25 (link) was maintained as on-feeder26 (link) or feeder-free27 (link) culture, as described previously.
10
Culturing MDCKII Cells Expressing Ovine ABCG2
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This study employed Madin-Darby Canine Kidney (MDCKII) cells that had been previously transduced with ovine ABCG2. The conditions for culturing have been previously described (32 (link)). Briefly, cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) enriched with glutamax (Life Technologies, Inc., Rockville, MD, United States) and supplemented with penicillin (50 units/mL), streptomycin (50 μg/mL), and 10% (v/v) fetal calf serum (MP Biomedicals, Solon, OH, United States) at a temperature of 37°C and 5% of CO2. Every 3 to 4 days, cells were trypsinized for subculturing.
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