The cells were seeded in a 35 mm dish and performed OGD/R and drug treatment. After 24 h of reoxygenation, replaced with fluorescent dye solution (DMEM: F-127: Fura-2 AM = 500: 1: 1) for 20 min, and then washed 3 times with artificial extracellular solution. Fluorescence of Fura-2 AM was measured at 340 nm (F340) and 380 nm (F380) under alternating excitation per second using an Olympus Digital Calcium Imaging System (IX73, DG-4PLUS/OF30, Japan). Images were acquired for 300s to obtain baseline Ca2+ levels with extracellular Ca2+ (1 mM). The extracellular solution was then replaced by adding BAPTA (1 mM, MedChemExpress, USA) or CaCl2 (Ca2+, 2 mM) and the ratio (F340/F380) was recorded. The △ratio (F340/F380) was calculated to indicate the change of [Ca2+]i levels before and after treatment with BAPTA or high calcium solution. The experiments were performed at least three independent experiments.
Bapta
Manufactured by MedChemExpress
Sourced in United States
BAPTA is a calcium chelator used in biochemical and cell biology research. It is a highly selective and potent calcium-binding agent that can be used to manipulate intracellular calcium levels. BAPTA has a high affinity for calcium ions and can be used to buffer or sequester calcium in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Xestospongin c, by Bio-Techne (1 mentions)
Ionomycin, by MedChemExpress (1 mentions)
Arachidonic acid, by MedChemExpress (1 mentions)
S trityl l cysteine stlc, by Santa Cruz Biotechnology (1 mentions)
Yoda1, by MedChemExpress (1 mentions)
Aacocf3, by Bio-Techne (1 mentions)
Blebbistatin, by Enzo Life Sciences (1 mentions)
Gsk205, by MedChemExpress (1 mentions)
Gsk1016790a, by MedChemExpress (1 mentions)
Bapta am, by MedChemExpress (1 mentions)
β actin mouse monoclonal antibodies, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Anti ha, by Proteintech (1 mentions)
Flag tag (flag), by Proteintech (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
P chk2, by Cell Signaling Technology (1 mentions)
4 protocols using bapta
1
Calcium Imaging in Ischemia-Reperfusion
2
Pharmacological Modulators of Cellular Mechanobiology
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The pharmacological and small‐molecule inhibitors and agonists used were the following: 7rh (2.5 µm ; Sigma) for DDR1 inhibition, rat anti‐human CD29 (1:100; BD Bioscience) to block integrin β1, blebbistatin (1 µm ; Enzo Life Sciences) for myosin II inhibition, GM6001 (10 µm ; Calbiochem) as broad‐spectrum MMP inhibitor, L‐mimosine (400 µm ; Santa Cruz Biotechnology) to arrest the cell cycle at G1, S‐trityl‐L‐cysteine (STLC; 20 µm ; Santa Cruz Biotechnology) to arrest at mitosis, ionomycin (10 µm ; MedChemExpress) to increase cell membrane permeability to calcium, gadolinium chloride (10 µm ; MedChemExpress) to inhibit mechanosensitive calcium channels, BAPTA (10 µm ; MedChemExpress) to chelate extracellular calcium, BAPTA‐AM (10 µm ; MedChemExpress) to chelate intracellular calcium, arachidonic acid (70 µm ; MedChemExpress) to mimic signaling downstream of nuclear mechanosensing, AACOCF3 (28 µm ; Tocris) to inhibit signaling downstream of nuclear mechanosensing, 2‐aminoethoxydiphenylborane (20 µm ; MedChemExpress) as IP3 receptor agonist, Xestospongin c (10 µm ; Tocris) as IP3 receptor agonist, GSMT×4 (10 µm ; MedChemExpress) as Piezo 1 inhibitor, Yoda1 (6 µm ; MedChemExpress) as Piezo 1 agonist, GSK205 (10 µm ; MedChemExpress) as TRPV4 inhibitor, and GSK1016790A (50 µm ; MedChemExpress) as TRPV4 agonist.
3
Antibody and Reagent Acquisition Protocol
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Anti-HA, FLAG, and β-actin mouse monoclonal antibodies were purchased from Proteintech (USA, Chicago), and an anti-S100A9 rabbit polyclonal antibody was purchased from Abcam (Cambridge, UK). BAPTA, a specific intracellular Ca2+ chelating agent, was purchased from MedChemExpress (New Jersey, USA). PMSF was purchased from Solarbio (Beijing, China). The anti-PRRSV N protein mAb was prepared in our lab.
4
Comprehensive DNA Damage Response Analysis
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The NO donor drug JS-K was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the Calcium ion chelating agent BAPTA was purchased from MedChemExpress (MCE, NJ, USA). JS-K and BAPTA were dissolved in dimethyl sulfoxide (DMSO) to concentrations of 5 mM and 20 mM respectively and stored in the refrigerator at -80 ℃. Antibodies against Phosphorylated histone H2AX (γH2AX), RAD51, PCNA, Phospho-ATM (p-ATM), Phospho-ATR (p-ATR), Phospho-Chk1 (p-Chk1), Phospho-Chk2 (p-Chk2), Phospho-Cdc2 (p-Cdc2), Cyclin B1, Phospho-CAMKKβ (p-CAMKKβ), Phospho-AMPKα (p-AMPKα), mTOR, Phospho-mTOR (p-mTOR), LC3B, Beclin 1, P62, GAPDH were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against AMPKα were purchased from Cohesion Biotechnology Co., Ltd (Beijing, China). The Horseradish peroxidase conjugated IgG secondary antibodies were obtained from Shanghai Beyotime Biotechnology Co., Ltd.
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