Both stable cell lines HUVEC‐AQP1 and HUVEC‐AQP1(T157A/T239A) were treated with hypotonic medium or hypertonic medium. Hypotonic medium was made by diluting DEME with deionized water (DMEM : H2O = 1 : 2). Hypertonic medium was ECGM2 containing 200 mm sorbitol. To assess the roles of PKC and calmodulin in the distribution and function of AQP1 protein, calmodulin antagonist W‐7 hydrochloride (Cat. No. 0369, Tocris, Minneapolis, MN, USA) and PKC inhibitor staurosporine (Cat. No. 1285, Tocris) were added into culture media while HUVEC‐AQP1 or HUVEC‐AQP1(T157A/T239A) cells were treated by osmotic stimuli. Five minutes, 10 min, and 15 min after osmotic treatment, the changes in AQP1 protein localization in and cell volume of HUVEC‐AQP1 or HUVEC‐AQP1(T157A/T239A) cells were assessed by fluorescence microscope.
Staurosporine
Manufactured by Bio-Techne
Sourced in United Kingdom
Staurosporine is a chemical compound that acts as a potent and broad-spectrum protein kinase inhibitor. It is commonly used in biological research as a tool to study various cellular processes involving protein phosphorylation.
Automatically generated - may contain errors
Lab products found in correlation
Thapsigargin, by Bio-Techne (3 mentions)
Arsenite, by Merck Group (2 mentions)
Trans isrib, by Bio-Techne (2 mentions)
Everolimus, by Bio-Techne (2 mentions)
Isrib, by Bio-Techne (2 mentions)
Mg132, by Enzo Life Sciences (2 mentions)
Pertussis toxin, by Merck Group (2 mentions)
Nps 2143, by Bio-Techne (2 mentions)
Baclofen, by Santa Cruz Biotechnology (2 mentions)
Carbachol, by Bio-Techne (2 mentions)
Saclofen, by Bio-Techne (2 mentions)
Skf81297, by Bio-Techne (2 mentions)
Pki 6 22, by Santa Cruz Biotechnology (2 mentions)
Pki 19 36, by Bio-Techne (2 mentions)
Jnj 16259685, by Santa Cruz Biotechnology (2 mentions)
Cinacalcet, by LGC (2 mentions)
Facsdiva software, by BD (2 mentions)
Chloroquine, by Bio-Techne (1 mentions)
Chloroquine diphosphate, by Bio-Techne (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
34 protocols using staurosporine
1
Osmotic Regulation of AQP1 in HUVECs
2
Treatment of Cellular Stress Pathways
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Arsenite (sodium Arsenite, Millipore Sigma, catalog no. S7400) was dissolved in water at 100 mmol/L, and the treatment was performed at a concentration of 300 μmol/L for 3 hours (50 (link)). MG132 (Enzo, catalog no. BML-PI102–0025) was dissolved in DMSO at 20 mmol/L, and the treatment was performed at a concentration of 10 μmol/L for 6 hours. ISRIB (trans-ISRIB, Tocris, catalog no. 5284) was dissolved in DMSO at 5 mmol/L and treated at a concentration of 1 μmol/L. Thapsigargin (Tocris, catalog no. 1138) was dissolved in DMSO at 5 mmol/L and treated at a concentration of 1 μmol/L for 12 hours unless otherwise indicated. Staurosporine (Tocris, catalog no. 1285), everolimus (Tocris, catalog no. 6188), and chloroquine (chloroquine diphosphate, Tocris, catalog no. 4109) were all dissolved in DMSO, and the treatment was performed at a concentration of 1 μmol/L, 5 μmol/L, and 100 μmol/L, respectively, for 12 hours.
3
Reagents and Antibodies for Cell Biology
4
Mitochondrial Dynamics Regulatory Agents
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Paraquat (MP Biomedicals, Santa Ana, CA), A23187, and ionomycin (Calbiochem/Merck, Darmstadt, Germany) were used at the indicated concentrations. Phorbol myristic acid (Calbiochem) was used at 1 μM. CCCP, antimycin A, valinomycin, and bafilomycin A1 were from Sigma-Aldrich (St. Louis, MO) and used at final concentrations of 20 μM, 40 μg/ml, 10 μM, and 100 nM, respectively. Staurosporine (Tocris Biosciences, Bristol, UK) was used at 2 μM. Mff and Mfn1 antibodies were described previously (Gandre-Babbe and van der Bliek, 2008 (link); Tanaka et al., 2010 (link)). The following antibodies were purchased: FLAG (Genescript, Piscataway, NJ), HA (Roche, Basel, Switzerland), Tom20, Tom40, Parkin, and normal mouse immunoglobulin G (IgG; Santa Cruz Biotechnology, Santa Cruz, CA), calnexin and Drp1 (BD Transduction Laboratories, Franklin Lakes, NJ), p62 (Abnova, Taipei City, Taiwan), porin (MitoSciences, Eugene, OR), LC3B (Sigma-Aldrich), Fis1 (Alexis Biochemicals and Proteintech, Farmingdale, NY), α-tubulin (Invitrogen, Carlsbad, CA), and actin (Sigma-Aldrich). Rabbit polyclonal antibodies were generated against polypeptide fragments of C. elegans DRP-1 protein and used along with other antibodies to probe blots of C. elegans extracts as described previously (Head et al., 2011 (link)).
5
Ex vivo and in vitro efferocytosis assays
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For the ex vivo assay, total PEC suspension was plated in a 24-well plate (1e6 cells/well) for 2 h in serum-free media. Cells were then washed 3 times with pre-warmed media before culturing overnight in complete RPMI media with either DMSO or 60uM pan-ACC inhibitor CP 640,186 (Sanbio, 17691-5). Thymocytes were isolated in the parallel to the PEC, by dissociating the thymus in a petri plate with frosted slides, and rendered apoptotic by overnight incubation with 2 µM staurosporine (Tocris, 1285). This typically yielded ~50% Annexin V+7AAD− cells. AC were labeled with cell-trace violet (ThermoFisher, C34557) according to the manufacturer’s instructions and then 1e6 cells were added directly to cultured macrophage for 60 min. AC were removed by consecutive washes with warm PBS before placing cells on ice in FACS buffer (2% FBS, 2 mM EDTA) to be harvest by scraping. For in vitro efferocytosis, a similar protocol was followed using jurkat cells and bone-marrow-derived macrophages cultured overnight with DMSO or ACC inhibitor.
6
Staurosporine-Induced BMDC Cytokine Profiling
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B16F0 cells were cultured and harvested as described above prior to treatment in vitro for 18 h with 0.25 µM staurosporine (Tocris Bioscience). staurosporine-treated cells were washed, resuspended in RPMI containing 10% FBS, and co-cultured at a 2:1 ratio (2 × 106 B16 cells:1 × 106 BMDCs) in 100 mm Petri dishes. After 24 h, biological replicates were pooled and BMDCs were enriched using a CD11c+ selection kit (Miltenyi Biotec) according to the manufacturer’s instructions. Enriched BMDCs were plated in RPMI supplemented with 10% FBS in 96-well round bottom plates at a density of 1.5 × 105 cells/well, and stimulated for 5 h with 50 ng/ml PMA plus 500 ng/ml ionomycin. Culture supernatants were collected and analyzed using the BioLegend LEGENDplexTM mouse inflammation cytokine panel kit according to the manufacturer’s instructions. In every experiment, at least 4 technical replicates were analyzed for each sample.
7
Electrophysiological Recordings with Diverse Pharmacological Agents
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Solutions were gravity-fed through a glass capillary (1.2 mm outer diameter) placed ~1 mm from the patch pipette tip. Most reagents were obtained from Sigma-Aldrich (Darmstadt, Germany). NPS 2143, PKI 19–36, staurosporine, saclofen, SKF 81297, carbachol, and CHPG were supplied by Tocris (Bristol, United Kingdom). PKI 6–22, JNJ 16259685, and baclofen were supplied by Santa Cruz Biotechnology (Dallas, United States). Pertussis toxin was supplied by Millipore Sigma (Burlington, Massachusetts). Cinacalcet was supplied by Toronto Research Chemicals (Toronto, Canada) and TTX by Alomone (Jerusalem, Israel). Phenytoin, carbamazepine, and CHPG were dissolved in DMSO (final concentration 0.125%). NPS 2143, calhex, MPEP, DMeOB, staurosporine, and chelerythrine chloride were dissolved in DMSO (final concentration ≤ 0.03%). JNJ 16259685 was dissolved in ethanol (final concentration 0.05%). Appropriate vehicle controls were performed for all experiments.
8
Annexin V and PI Staining for Apoptosis
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Cells were plated in DMEM in 6-well plates in triplicate in the presence of 2-mercaptoethanol, 1X MEM NEAA mixture, 10% FBS, and doxycycline (1 μg/mL). Twenty-four hours after plating, cells were washed twice with PBS and media was changed to DMEM with 10% dialyzed FBS and doxycycline (1 μg/mL). Seventy-two hours later, cells were detached with Accumax (Sigma-Aldrich, A7089) and washed twice with cold PBS on ice. Cells were stained with annexin V and propidium iodide (PI) according to the manufacturer’s instruction (BD, 556547). Samples were analyzed using an LSRFortessa (BD) flow cytometer, and the fractions of stained cells were quantified using FloJo 10.6, with Staurosporine (4 hr, 5 μM) (Tocris, 1285) used as a positive control for cell death and to help establish gating of the sorted cells.
9
Inducing Cell Death in FIECs and HT3B3 Cells
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Death was induced in FIECs by incubation with 1 µm z-VAD-fmk and 1 µm staurosporine (TOCRIS Bioscience) in DMEM plus 20% FBS at 37 °C in 5% CO2 for 8 h59 (link). FIECs were stained with 500 nm PI and 2 µg/ml H33342 for 5 min in the dark prior to visualization by confocal microscopy.
RIP1K-dependent necroptosis was induced in HT3B3-WT and HT3B3-KO cells using by pretreating cells with 100 nm BV6, 500 nm qVD-OPh, and 1 µm cyclohexamide at 37 °C in 5% CO2125 (link). After 1 h incubation, human TNFα was added to a final concentration of 10 ng/ml and incubated for 12–16 h at 37 °C in 5% CO2. Cell death was measured by uptake of 100 nm Sytox™ green imaged on the BioTek Cytation™ 5.
RIP1K-dependent necroptosis was induced in HT3B3-WT and HT3B3-KO cells using by pretreating cells with 100 n
10
Assessing Apoptosis in Mouse Neutrophils
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Apoptosis in isolated mouse bone marrow neutrophils was assessed by FACS analysis using Annexin V-FITC kit (Miltenyi Biotec, Madrid, Spain) after incubation with staurosporine (200 nM; Tocris Bioscience, Bristol, UK) or human postprandial TRLs (100 μg of TGs/mL) (see below) for 12 h. Apoptotic cells were labelled with Annexin V conjugated to green-fluorescent Alexa Fluor 488 dye and necrotic cells were labelled with red-fluorescent propidium iodide (PI) (Thermo Fisher Scientific, Madrid, Spain). These populations were evaluated using FACSCanto II Cell Analyser (Becton Dickinson, Madrid, Spain) with an excitation wavelength of 488 nm and a 530 nm filter for the detection of Alexa Fluor 488 and a 585 nm filter for the detection of PI. The data were analysed using FACSDiva software (Becton Dickinson, Madrid, Spain). At least 10,000 events for each sample were analysed and gated according to light scattering properties.
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