PMC:1134658 / 22781-29525 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1134658","sourcedb":"PMC","sourceid":"1134658","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1134658","text":"Methods\n\nCell culture\nIf not specified, all cells were cultured in X-VIVO 15™ (BioWhittaker, Verviers, Belgium) serum-free medium at 37°C and 5% CO2 in air.\nPeripheral blood was provided by the Blood Bank at Buskerud Regional Hospital with formal agreement by the patients, and approval by the regional ethics committee. Highly purified resting human B-lymphocytes (CD19+ cells) were isolated from the peripheral blood by rosetting with immunomagnetic beads (Dynabeads M450; Dynal, Oslo, Norway) as described [55]. This procedure yields less than 0.5% T cells, 0.1% NK cells, and 0.5% monocytes as judged by indirect immunofluorescence staining.\nThe following cell lines from human lymphoid malignancies were maintained in RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% foetal bovine serum (FCS), 100 units/ml penicillin G, and 100 units/ml of streptomycin sulphate, but serum-starved for at least four hours and cultured in X-VIVO 15™ when included in experiments: EBV-negative BL cell lines Ramos (ECACC 85030802), HL60 (JCRB0085).\n\nGrowth factors/supplements\nThe following reagents were used at indicated concentrations: recombinant human (rhu) BMP-6 (1 μg/ml, if not specified otherwise), rhu BMP-RIB/ALK-6/Fc Chimera (5 μg/ml), rhu BMPR-II/Fc Chimera (5 μg/ml), and recombinant mouse Noggin (5 μg/ml) were purchased from R\u0026D Systems (Abingdon, UK); Anti-IgM F(ab)2 fragments of rabbit polyclonal antibodies to human IgM heavy chain (37.5 μg/ml) was obtained from Dako, Copenhagen, Denmark and rhu CD40 ligand (CD40L, 10 ng/ml) was a gift from Immunex Corp. (Seattle, WA).\n\nAntibodies used for flow cytometric analysis and immunoblot analysis\nAntibodies against the human BMP-receptors Act-RIA, BMP-RIB, BMPR-II, Act-RIIA and Act-RIIb were purchased from R \u0026 D Systems (Abingdon, UK). Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R \u0026 D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).\nCharacterisation of BMP-signalling pathways was done by use of anti-phospho-Smad1, -5, -and 8 polyclonal antibody (Chemicon, Temecula, CA, USA). Expression levels of Id1–3 proteins were detected with polyclonal rabbit antibody and detection was blocked with blocking peptide from Santa Cruz Biotechnology (Santa Cruz, San Diego, CA, USA). As secondary antibodies served anti-mouse, anti-goat or anti-rabbit IgG- horseradish peroxidase (HRP) from Dakocytomation AS (Copenhagen, Denmark) for immunoblot analysis. Anti-β-actin was from Santa-Cruz. From Becton Dickinson (San Jose, CA), we purchased anti-CD19-PE, anti-CD19-FITC. The antibodies used for cell sorting were anti-CD19 PC5 from Immunotech SA (Marseille, France) and anti-CD27 PE from Becton Dickinson, Biosciences Pharmingen (San Diego, CA, USA).\n\nCell sorting\nHighly purified CD19+CD27- or CD19+CD27+ cells were obtained by staining CD19+ cells with anti-CD27 PE and CD19 PC5 mAbs for 30 minutes at 4°C, followed by washing with PBS and sorting on FACS DiVa from Becton Dickinson.\n\nWestern-blot analysis\nB cells from peripheral blood or cultured cell-lines were lysed in lysis buffer (glycerol 10%, β-mercaptoethanol 5%, 0.0625 M Tris-HCL [pH 6.8], sodium dodecyl sulphate [SDS] 2.5%w/vol). Total protein (30–100 μg) from each sample was run on 10% or 12% SDS/polyacrylamide (SDS/PAGE) gels and blotted onto nitrocellulose filters (Protran; Schleicher \u0026Schuell GmbH, Dassel, Germany). Blocking, washing and incubation of the filters with primary antibodies were done according to the manufacturer's protocols at room temperature (RT). After washing with TBS/0.1% Tween-20 (TBS-T), the filters were incubated with horseradish peroxidase (HRP) coupled to relevant secondary antibodies (see above) for 60 minutes at RT. Enzyme activity was visualised by the enhanced chemiluminescence system, ECL+PLUS (Amersham, Buckinghamshire, UK). Densitometric analysis was performed by scanning hyperfilms on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). Quantification of Id1, Id2 and Id3 protein was calculated by normalizing the specific protein bands to β-actin using Image Quant 5.5 software (Molecular Dynamics).\n\nAnalysis of BMP-6 messenger RNA (mRNA) expression\nEndogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. Total RNA was isolated using Absolutely RNA™ RT-PCR Miniprep Kit (Stratagene Europe, Amsterdam, Netherland) according to the manufacturers instructions. Quantification of the isolated total RNA was achieved by using spectrophotometric OD260 measurements. Equal amounts of RNA were then reverse transcribed to cDNA with TaqMan® Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, U.S.A). To measure mRNA expression of BMP6, Id1–Id4 and PGK1 PCR were carried out with TaqMan® universal master mix. Primers and probes were provided by Assay-on-Demand (Applied Biosystems). PCR reactions were carried out in a final volume of 25 μl (BMP-6) or 20 μl (ID1). The cDNA added to each reaction was equivalent to the input of 20 ng of total RNA. The gene expression was quantified using the standard curve method (BMP6), or the comparative CT method (Id1) as described in ABI7700 User Bulletin 2 (Applied Biosystems). The expression was then normalized to the expression level of PGK1. PGK1 was chosen, because it has been shown to have low expression variability among lymphocyte specimens [56]. Expression levels in B cells were then related to the expression levels in Ramos cells.\n\nCell proliferation\nFor estimation of DNA synthesis, CD19+ cells (7.5 × 104 cells/0.2 ml) or Ramos cells (1 × 104 cells/0.2 ml) were cultured in triplicate in microtiter wells. The cells were pulsed with 3.7 × 104Bq [3H]thymidine (Amersham, Buckinghamshire, UK) for the last 16 h of a 72-h incubation. The cells were harvested using an automated cell harvester (Packard Instrument Company, Meriden, CT, USA) and [3H]thymidine incorporation was determined in a scintillation counter (TopCount, Packard Instrument Company Inc., Meriden, CT).\n\nDetermination of cell death\nCell death was measured by vital dye exclusion test by staining cells with 5 μg/ml propidium iodide ([PI]; Calbiochem Corp.; La Jolla, CA; 5 mg/ml) for one minute on ice. At least 1,000 cells per sample were run on a BD FACSCalibur flow cytometer.\n\nStatistical analysis\nThe statistical significance of differences between groups was determined using the paired two-tailed Wilcoxon nonparametric test, by applying SPSS10.1 software (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered 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