The ROCK inhibitor Y-27632 (Sigma, Y-0503) and cytochalasin B (Sigma, C-6762) were diluted in DMSO as a 100mM stocks rand were added to larvae at a concentration of 100µM. The sphingosine kinase inhibitor SKI 5C (Sigma, S8326) was diluted in DMSO as a 10mM stock and was added to larvae at a concentration of 20µ M. The Apoptosis Inhibitor (AI) (Santa Cruz, NS3694) was diluted in DMSO as a 10mM stock and was added to larvae at a concentration of 2µM. The caspase inhibitor zDEVD-fmk (Sigma, CAS210344–95–9) was diluted in DMSO as a 10mM stock and was added to larvae at a concentration of 10µM. 25 mg of BAPTA (Invitrogen; B1204) were diluted in 10mL of E3 to achieve a final concentration of 10mM. Except for BAPTA that was only added after mounting larvae in agarose, larvae were treated with the inhibitors simultaneously as MTZ, and fresh inhibitor was added after MTZ washout in both agarose and E3 for live-imaging experiments and in E3 for immunofluorescence assays. Of note, laser ablations and analyses of pulses and extrusions were performed throughout the 2h window following MTZ treatment during which cell-cell junctions and tissue integrity are not compromised, and extrusion events occur following typical rosette pattern formation
Bapta
Manufactured by Thermo Fisher Scientific
Sourced in United States, Belgium
BAPTA is a chemical compound that functions as a calcium chelator. It is commonly used in biochemical and cell biology research applications to control and manipulate the concentration of free calcium ions within experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Cgp55845, by Bio-Techne (8 mentions)
Picrotoxin, by Abcam (6 mentions)
Sch23390, by Bio-Techne (6 mentions)
Sulpiride, by Bio-Techne (5 mentions)
K methylsulfate, by Thermo Fisher Scientific (5 mentions)
Scopolamine hydrobromide, by Bio-Techne (4 mentions)
Mk 801, by Abcam (4 mentions)
K methylsulphate, by Thermo Fisher Scientific (3 mentions)
Egta am, by Merck Group (3 mentions)
Bapta am, by Thermo Fisher Scientific (3 mentions)
Histamine, by Merck Group (2 mentions)
Qx 314, by Bio-Techne (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
4 aminopyridine, by Merck Group (2 mentions)
Bapta am, by Merck Group (2 mentions)
α methyl dl tyrosine, by Merck Group (2 mentions)
Scopolamine hydrobromide, by Merck Group (2 mentions)
Sch 23390 hydrochloride, by Bio-Techne (2 mentions)
ω agatoxin tk, by Alomone (2 mentions)
Dihydro β erythroidine hydrobromide, by Bio-Techne (2 mentions)
47 protocols using bapta
1
Modulation of Cell Extrusion Dynamics
2
Zebrafish Hair Cell Endocytosis Assay
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Long-term dye incorporation experiments were performed as described previously (56 (link)). Briefly, 6 dpf zebrafish larvae were pre-incubated for 10 min in BAPTA (Thermo Fisher Scientific) to disrupt the tip links and thus block FM1–43 entrance through the mechanotransduction channels, and then for 90 min with 3 μm of FM1–43FX (Thermo Fisher Scientific’) in the presence of low BAPTA concentrations for assessment of vesicle endocytosis. After several washes, animals were fixed with 4% PFA, counterstained with phalloidin and processed for fluorescence analyses. Fluorescence intensity was estimated using ImageJ as previously described (56 (link)).
3
Antibody-Dependent Cellular Cytotoxicity Assay
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All antibody-dependent cellular assays were performed in the presence or absence of 5 µg/mL cetuximab (anti-EGFR, Merck KGaA) or trastuzumab (anti-HER2/neu, Roche) and with or without addition of 5 µg/mL SIRPα blocking antibody (clone 12C44 (link)). For the experiments with or without extracellular Ca2+, neutrophils and tumor cells were taken up in HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) supplemented with 5 g/L human albumin (Albuman, Sanquin Plasma Products) and 5.5 mM glucose or Minimal Essential Medium (MEM, Gibco), both with or without 1 mM CaCl2. For the experiments with the cell permeant chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N, N, N’, N’-tetraacetic acid), tumor cells were pretreated with BAPTA (ThermoFisher) for 30 min at 37°C and washed before coincubation with neutrophils. For the MEMbrane-cholesterol depletion experiments, tumor cells were preincubated with 2.5 mM methyl-β-cyclodextrin (MβCD) (Sigma) for 20 min at 37°C and washed before coincubation with neutrophils.
4
Intracellular Calcium Measurement
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Restriction and modification enzymes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Digitonin, histamine, CPA, DMEM, trypsin, L-glutamine, and penicillin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). BAPTA and Fura-2 were purchased from Invitrogen, Thermo Fisher Scientific (Carlsbad, CA, USA). All other materials were analytical or of the highest available grade.
5
Pharmacological Manipulation of Neuronal Activity
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Picrotoxin was obtained from Abcam. QX314, NBQX, and AP5 were obtained from Tocris. BAPTA was from Invitrogen. All other chemicals were from Sigma-Aldrich.
6
Neurotransmitter Modulation Reagents
7
Modulation of Autophagy Flux in IPC
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PC12 cells or cortical neurons were pretreated with dibromo-1,2-bis(aminophenoxy) ethane N,N,N9,N9- tetraacetic acid (BAPTA, Invitrogen B1205) 0.125 μM, 0.5 μM, 2 μM or compound C (Dorsomorphin, Selleck S7306) 5 μM 60 min before IPC. The cells continued to be incubated with these agents during the reperfusion episode after IPC. To access autophagy flux, ammonium chloride (NH4Cl), an inhibitor of lysosome acidification, was used to inhibit lysosome degradation. PC12 cells were treated with or without NH4Cl (Greagent G17391B) 20 mM for 12 h during the reperfusion episode after IPC.
8
Dendritic Cell Maturation and Calcium Signaling
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Micro-channels were coated with fibronectin (Sigma) or PLL(20)-g[3.5]-PEG(2) (SuSoS Chemical). For myosin light chain kinase inhibition, cells were incubated with different concentrations of ML7 from Calbiochem as indicated for 16 h. For Ca2+ experiments, Oregon Green BAPTA 1-AM, FuraRed, BAPTA (Invitrogen), and Thapsigargin from Calbiochem were used. For IP3R inhibition, 5 μM xestospongin C from Calbiochem was used. For dendritic cell maturation, we incubate the cells 24 h with 100 ng/ml LPS (Sigma). For flow cytometry analysis, we used a homemade 24G2 anti-Fc Receptor antibodies, rabbit serum from Agro Bio as a control, and anti-CD11c (HL3 clone), anti-IAbb (AF6-120.1 clone), and anti-CD86 (GL1 clone). For immunoblot, we used anti-IP3R type 1 (Abcam ab5804), anti-IP3R type 3 (610313 BD Transduction Laboratories), anti-phospho-myosin light chain (Rockland 600-401-416), and anti-actin (Millipore). For lentivirus production, HEK cells were transfected using GeneJuice (Novagen).
9
Ba2+ Currents Measurement Protocol
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The bath solution used for recording Ba2+ current contained 150 mM NaCl, 10 mM BaCl2-2H2O, 1 mM MgCl2, 10 mM HEPES, and 8 mM glucose and was titrated to pH 7.4 with NaOH. The pipette solution contained 160 mM CsCl, 5 mM MgCl2, 5 mM HEPES, 0.1 mM 1,2-bis(2-aminophenoxy)ethane N,N,N′N′-tetraacetic acid (BAPTA), 3 mM Na2ATP, and 0.1 mM Na3GTP and was titrated to pH 7.4 with CsOH. Reagents used were oxotremorine-M (Oxo-M; Research Biochemicals); BAPTA, DMEM, FBS, Lipofectamine 2000, and penicillin/streptomycin (Invitrogen); and ATP, GTP, and other chemicals (Sigma-Aldrich).
10
Purinergic Receptor Inhibition Assay
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The following drugs were used: BAPTA (30 mM; Invitrogen, Carlsbad, CA, USA), 6-N, N-Diethyl-D -β,γ-dibromomethylene ATP trisodium salt (ARL 67156 trisodium salt, 50 μM; Tocris Bioscience, UK), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 5 μM; Tocris Bioscience, UK).
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