For measurements of intracellular free calcium concentration ([Ca2+]i), RBC-depleted single-cell suspended splenocytes were simultaneously stained with CD8-PE (53–6.7; BD Biosciences), CD4-APC (GK1.5; BioLegend) or B220-PE (RA3–6B2; BioLegend) and loaded with Indo-1 acetoxymethyl (Indo1-AM; Molecular Probes), as described previously (8 (link)). For analysis of ([Ca2+]i), cells were suspended at 10×106 cells/ml in warm IMDM + 2% FBS in a 500ul volume. Cells were acquired for 30 seconds to establish a baseline, and then stimulated with 5μg/ml of F(ab’)2 rabbit anti-mouse anti-IgG (H&L; Invitrogen) +/− indicated doses of Idelalisib (LC Laboratories) and acquired for 3 minutes. For CD4 and CD8 T cells, cells were acquired for 30 seconds to establish a baseline, and then 10μg/ml of anti-CD3-biotin (145–2C11; BD Biosciences) +/− indicated doses of Idelalisib was added, 60 seconds later 20μg/ml streptavidin (Sigma-Aldrich) was added and acquired for 3 minutes. Mean relative ([Ca2+]i) was monitored over time using an LSR Fortessa X-20 (BD) with analysis using FlowJo software (Tree Star).
Idelalisib
Manufactured by Merck Group
Sourced in Spain
Idelalisib is a laboratory product manufactured by Merck Group. It is a small molecule that inhibits the delta isoform of phosphoinositide 3-kinase (PI3Kδ). The core function of Idelalisib is to serve as a research tool for investigating cellular signaling pathways involving PI3Kδ.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Indo 1 acetoxymethyl indo1 am, by Thermo Fisher Scientific (1 mentions)
Cd4 apc gk1.5, by BioLegend (1 mentions)
F ab 2 rabbit anti mouse anti igg h l, by Thermo Fisher Scientific (1 mentions)
Anti cd3 biotin 145 2c11, by BD (1 mentions)
Idelalisib, by LC Laboratories (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Cd8 pe, by BD (1 mentions)
Acetylsalicylic acid asa, by Merck Group (1 mentions)
Sdf 1α, by Thermo Fisher Scientific (1 mentions)
Anti cd19 fitc antibody, by BD (1 mentions)
α igm f ab 2, by Southern Biotech (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
3 protocols using idelalisib
1
Measuring Intracellular Calcium in Splenocytes
2
Idelalisib and Antiplatelet Drugs in Thrombosis
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C57BL/6J mice (Envigo Rms Spain Sl., Sant Feliu de Codines, Spain) were kept under controlled environmental conditions (relative humidity: 45–65%; temperature: 20–24 °C) with a 12 h light/dark cycle and free access to chow and water. At the time of the study, mice were 8–12 weeks old. Equivalent numbers of males and females were included. According to the oral drug administration protocol, mice were immobilized and Idelalisib, 20 mg/kg or vehicle (DMSO; Sigma-Aldrich, Madrid, Spain), was introduced directly into the stomach with an oral gavage feeding tube. One hour after the treatment, tail bleeding and arterial thrombosis assays were performed. A separate set of animals treated with other established antiplatelet drugs were also used as positive controls. Acetyl salicylic acid (ASA; Sigma-Aldrich, Madrid, Spain) was dissolved in water at 1 mg/mL and given by a single intraperitoneally injection of 100 mg/kg in mice 2–4 h before the experiment, as previously reported [33 (link)]. Moreover, two doses of clopidogrel (2 mg/kg) were also given to both animal groups by oral gavage 24 h and 2 h before the experiment.
3
Investigating PI3K Inhibitor Effects in B Cells
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PI3K inhibitors CZC24832, GS-1101/idelalisib, IPI-145/duvelisib, and GDC-0980/apitolisib (Selleck Chemicals) were reconstituted in DMSO (Sigma) and used at final concentrations of 2 μM (CZC24832) and 1 μM (idelalisib, duvelisib, GDC-0980). CZC24832 has greater than 10-fold selectivity over PI3Kβ and greater than 100-fold selectivity over PI3Kα and PI3Kδ [33 (link)]. α-IgM F(ab′)2 (Southern Biotech) was used at 10 μg/ml and CD40 ligand and interleukin 4 (R&D systems) were used at 50 ng/ml each. Anti-p110γ antibody, anti-Akt antibody, anti-pAkt Ser473 antibody, anti-GAPDH antibody (Cell Signaling), anti-p101 antibody (R&D systems), and Mini-PROTEAN TGX precast gels (Bio-Rad) were used for Western blot analysis. SDF1α (Peprotech) was used at 100 ng/ml. Alexa Fluor® 488 Phalloidin (Life Technologies) was used at 33 nM for F-actin staining. Superscript VILO cDNA mix (Invitrogen) was used for RT-qPCR. DAPI, trypsin-EDTA (Sigma), and anti CD19-FITC antibody (BD Biosciences) were used for the cell adhesion assay.
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