Cells were cultured in serum-free medium (SFM) for 5 (OCI-Ly10) or 2 (HKBML) days, according to their sensitivity to serum starvation. Starved OCI-Ly10 (1 × 105 cells/well) or HKBML cells (2 × 105 cells/well) were cultured for 3 days in the presence of 500 ng/ml APRIL or BAFF protein (both R&D Systems, Minneapolis, USA) under SFM conditions at 37 °C in a humidified atmosphere (5% CO2 in air). Binding of APRIL and BAFF was blocked with 1 μg/ml TACI-Fc (R&D Systems, Minneapolis, USA) by pre-incubation for 30 min. OCI-Ly10 and HKBML cells (5 × 104 cells/well) were treated with MTX (50 nM) in absence or presence of APRIL or BAFF. Blocking of pro-survival effects was performed by adding TACI-Fc (1 μg/ml). Cells were cultured for 3 days at 37 °C and 5% CO2 in a humidified atmosphere. All experiments were repeated at least 3 times.
April
Manufactured by R&D Systems
Sourced in United States
APRIL is a laboratory instrument used for the analysis of biological samples. It is designed to perform various assays and measurements that are essential for research and development in fields such as immunology, cell biology, and biochemistry. The core function of APRIL is to provide accurate and reliable data to support scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
April, by Thermo Fisher Scientific (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Recombinant baff, by R&D Systems (1 mentions)
Lps from e coli 0111 b4, by Merck Group (1 mentions)
Cyclohexamide, by Merck Group (1 mentions)
Unconjugated goat anti mouse igm f ab 2, by Jackson ImmunoResearch (1 mentions)
Cpg oligonucleotides odn 1826, by InvivoGen (1 mentions)
Magnisort mouse f4 80 positive selection kit, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse baff, by R&D Systems (1 mentions)
3t3 l1 preadipocyte, by American Type Culture Collection (1 mentions)
Live dead kit, by Thermo Fisher Scientific (1 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
Easysep human b cell enrichment kit, by STEMCELL (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Torin 1, by Cell Signaling Technology (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Il 10, by BioLegend (1 mentions)
2 mercaptoethanol βme, by Merck Group (1 mentions)
Recombinant tnf, by R&D Systems (1 mentions)
9 protocols using april
1
APRIL and BAFF Modulate MTX Response
2
Apoptosis in CLL Cells: BAFF/APRIL Effects
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PB mononuclear cells from six patients with CLL, exhibiting high (>40%, 3 patients) or very low to absent (<10%, 3 patients) TACI expression, were isolated by density gradient centrifugation and diluted with Iscove's Modified Dulbecco's Medium (IMDM, Life Technologies) supplemented with 10% fetal bovine serum (FBS), at a concentration of 8 × 105 cells/mL. Afterwards, aliquots of 0.5 mL of cells were plated on a 96-well plate and stimulated by either 1 μg/mL of recombinant BAFF (R&D Systems, Mineapolis, USA), or 200 ng/mL APRIL (R&D Systems), or combinations of the above, diluted into 0.1 mL IMDM. Another aliquot of 0.5 mL of cells supplemented with 0.1 mL IMDM without any stimulus was used as internal control. Samples were then incubated at 37°C in the presence of 10% CO2 for 24 h. Subsequently, apoptosis was measured by flow cytometry using an Annexin V-FITC/7-AAD (7-AAD) kit (BC), according to the manufacturer's instructions.
3
Isolation and Stimulation of Mouse B Cells
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Mouse B cells were isolated by the EasySep Mouse B cell Negative selection Kit (STEMCELL Technologies, Vancouver) according to the manufacturer's protocol. Purity based on CD19 expression was around 95–97% as determined by flow cytometry. Cells were seeded at a cell density of 2.5 x 105 or 3 x 106 mL−1, for flow cytometry staining and ELISA, and RNA extraction, respectively. Cells were stimulated in complete RPMI with 10 μg mL−1 of unconjugated goat anti‐mouse IgM F(ab’)2 (Jackson ImmunoResearch Laboratories, West Grove, PA), 10 μg mL−1 or 100 ng mL−1 lipopolysaccharide (LPS) from E. coli 0111:B4 (Sigma‐Aldrich), 1 μg mL−1 APRIL (R&D Systems, Minneapolis, MN), 1 μg mL−1 BAFF (R&D Systems), or 1 μg mL−1 CpG oligonucleotides ODN 1826 (InvivoGen, San Diego, CA). To induce IkBα degradation, 1 x 106 cells were stimulated in 100 μL complete RPMI medium for 90 min with 10 μg mL−1 of unconjugated goat anti‐mouse IgM F(ab’)2 (Jackson ImmunoResearch Laboratories), 50 ng mL−1 PMA and 1 μm ionomycin, or 0.1 μm calyculin A, in the presence of 10 μm cyclohexamide (all from Sigma‐Aldrich).
4
Isolation and Culture of Macrophages and Adipocytes
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For isolation of peritoneal Mϕs, peritoneal exudate cells were harvested and F4/80+ cells were purified using the MagniSort Mouse F4/80 Positive Selection Kit (eBioscience). For VAT Mϕ isolation, SVCs were firstly separated with 30 and 70% Percoll gradient and VAT Mϕs were isolated using the F4/80 positive selection kit. Conditioned media (CM) were collected from Mϕs cultured in DMEM/F12 medium for 2 days. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) and freshly isolated SAT mesenchymal cells were cultured in DMEM. The differentiation and staining of adipocytes were performed as described by Alexaki et al. (11 (link)). For BAFF and APRIL stimulation, 100 ng/mL recombinant mouse BAFF (R&D Systems, Minneapolis, MN) or APRIL (Peprotech, Rocky Hill, NJ) was added into differentiation and maintenance medium.
5
Characterizing Memory B Cell Responses
6
Isolation and Stimulation of Mouse and Human B Cells
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Mouse splenic resting B cells were negatively isolated with magnetic beads20 (link),58 (link) (Cat. 130-090-862, Miltenyi Biotec), whereas human follicular and MZ B cells were FACSorted as further detailed below. Human splenic total IgD+ B cells were MACSorted. Briefly, splenocytes were first incubated with biotin-conjugated anti-IgD antibody (Supplementary Table 2 ) and then anti-biotin beads. Each labeling step included 15-min incubation at 4 °C. Magnetic column-based selections were performed and resulting positive fracion was isolated (Miltenyi Biotec). Splenocytes, 2E2 B cells and human embryonic kidney 293 cells were kept in culture as already reported4 (link),56 (link). Human MZ, naive, or IgD+ B cells were incubated with 500 ng/ml APRIL MegaLigand (Alexis) with or without 0.1 μg/ml CpG (Invitrogen). When indicated, 10 nM rapamycin (Sigma), 10 nM Torin 1 (Cell Signaling), or 25 μM Ly294002 (Calbiochem) were added 30 min before treatment with APRIL and/or CpG. Mouse B cells were stimulated with 100 ng/ml APRIL (R&D) in the presence or absence of 0.5 μg/ml LPS (Sigma-Aldrich).
7
BAFF and APRIL Cytokine Levels and Immunoglobulin Assessment
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Serum levels of BAFF and APRIL cytokines were determined at baseline and at specific time points during follow-up using BAFF (R&D Systems, UK) and APRIL (Invitrogen/Life Technologies, UK) ELISA kits following the manufacturers’ instructions. Samples were run in duplicates. For the BAFF and APRIL ELISA analyses, sera from 56 or 22 healthy blood donors, respectively, were included for comparison. The plates were read using a FLUOstar Omega plate reader (BMG Labtech, Germany). Serum BAFF and APRIL levels were quantified using the in-kit standard curves for the respective cytokines. To adjust for technical variations in measurements of absolute BAFF concentrations between plates, all samples were calibrated using a reference plate containing several samples from all other plates.
Serum immunoglobulin levels (IgG, IgA, and IgM) were measured for all time points up to 24 months after intervention for patients included in KTS-2-2010 trial using a Siemens BN ProSpec Nephelometer (Erlangen, Germany).
Serum immunoglobulin levels (IgG, IgA, and IgM) were measured for all time points up to 24 months after intervention for patients included in KTS-2-2010 trial using a Siemens BN ProSpec Nephelometer (Erlangen, Germany).
8
Plasma Cytokine Quantification by ELISA
9
Thymocyte Culture and NF-κB Activation
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Thymocytes were cultured at 37°C with 5% CO2 in RPMI-1640 (Invitrogen) supplemented with 10% (vol/vol) FBS (Invitrogen), 0.1% (vol/vol) 2-mercaptoethanol βME (Sigma-Aldrich), and 1% (vol/vol) penicillin-streptomycin (Invitrogen; RPMI-10). Recombinant TNF, BAFF, LIGHT, APRIL, TRAIL, GITRL, CD70, and TLA1 were supplemented to cultures at 100 ng/ml unless otherwise state, and were obtained from R&D Systems, with PBS used as vehicle. IKK2 inhibitor Bl605906 was used at 10 µM in DMSO vehicle. Binding of RelA from nuclear extracts of TNF-stimulated thymocytes to NF-κB oligonucleotide was determined by specific ELISA (Active Motif) according to the manufacturer’s instructions.
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