To assess senescence status, we used a β-gal imaging method. In brief, cells were split into 12-well dishes (0.125 × 106 cells per well) with a glass cover slide at the bottom of each well and allowed to settle for 24 hours. Cells were first pretreated with bafilomycin A1 (Selleckchem, S1413, 622.83 g/mol, 100 μM stock). Existing medium was aspirated, and then cells were washed with phosphate-buffered saline (PBS) and replaced with treated bafilomycin A1 media for 30 min at a final concentration of 100 nM. Following bafilomycin A1 pretreatment to normalize lysosome activity, C12FDG (Invitrogen, #D2893, 853.92 g/mol, 10 mM stock) was added directly to the existing media for 90 min at a final concentration of 10 μM. Note that because of light sensitivity, exchange was conducted in a dark environment. Following bafilomycin A1 and C12FDG treatments, medium was aspirated, and cells were washed with PBS three times, fixed with 4% paraformaldehyde/PBS (10 min), followed by two PBS washes, and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, #P36935) and mounted onto coverslips. Fixed cells were immediately imaged using a ZOE fluorescent cell imager (Bio-Rad). Percent cell positively was calculated using ImageJ with a background/negative threshold value. Any cells above this threshold were considered positive. All cells in each image frame were counted.
Bafilomycin a1
Manufactured by Thermo Fisher Scientific
Sourced in United States, New Zealand
Bafilomycin A1 is a macrolide compound that functions as a selective and potent inhibitor of vacuolar-type H+-ATPase (V-ATPase). V-ATPase is an enzyme that plays a crucial role in regulating intracellular pH and ion homeostasis in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
C12fdg, by Thermo Fisher Scientific (3 mentions)
Bafilomycin a1, by Selleck Chemicals (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Chicken anti rabbit alexa 594, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Anti hmga2 and p21, by Cell Signaling Technology (2 mentions)
Alexa 488 chicken anti mouse, by Thermo Fisher Scientific (2 mentions)
α tubulin, by Abcam (2 mentions)
Anti h3k9me3, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Zoe fluorescent cell imager, by Bio-Rad (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Bam15, by Cayman Chemical (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Haloperidol, by Merck Group (1 mentions)
Fluoxetine hydrochloride, by Merck Group (1 mentions)
Ketamine hydrochloride, by Merck Group (1 mentions)
Scopolamine hydrobromide, by Merck Group (1 mentions)
30 protocols using bafilomycin a1
1
Senescence Assessment via β-Gal Imaging
2
Anti-CD20 mAb Mediated Cell Death
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Anti-CD20 mAb (clone 5D2, mouse IgG2a, from Genentech) was used at 20 μg ml−1 in vitro. Mice were injected intravenously with 50 μg of 5D2 or 50 μg of the isotype control HY1.2 (mIgG2a) when indicated. In some experiments, cells were pretreated for 1 hour with bafilomycin A1 (Fisher Scientific) at 50 mM before anti-CD20 mAb addition, and bafilomycin A1 was left throughout the assay. Tumors were treated overnight with staurosporine (Sigma-Aldrich) at a final concentration of 1 μM to induce apoptosis. Mice received 200 μl of rituximab, an anti-human CD20 IgG1 Ab, when indicated.
3
Bafilomycin A1 and Chloroquine Treatment Protocols
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For bafilomycin A1 treatment, semi-intact hearts were incubated with 100 nM of bafilomycin A1 (Fisher Scientific, AAJ61835MCR) in artificial hemolymph (buffer receipt in [22 (link)]) for 2 h at room temperature prior to the appropriate immunostaining. DMSO was used as a control.
For chloroquine treatments, 100 µl of 20 mM chloroquine diphosphate salt, CQ (Fisher Scientific, ICN19391925) was added onto the fly food. Flies were fed with chloroquine for at least 24 h prior to the cardiac analysis or the western blots. To test the toxicity of CQ treatment, we performed a survival analysis by feeding ywR flies with 20 mM CQ and observed the mortality within a period of within two weeks (about 100 flies per treatment). Flies were transferred to fresh CQ food every day and fly mortality was recorded daily.
For chloroquine treatments, 100 µl of 20 mM chloroquine diphosphate salt, CQ (Fisher Scientific, ICN19391925) was added onto the fly food. Flies were fed with chloroquine for at least 24 h prior to the cardiac analysis or the western blots. To test the toxicity of CQ treatment, we performed a survival analysis by feeding ywR flies with 20 mM CQ and observed the mortality within a period of within two weeks (about 100 flies per treatment). Flies were transferred to fresh CQ food every day and fly mortality was recorded daily.
4
Evaluating Mitochondrial Function in Liver Cells
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HepG2 and primary human hepatocytes were plated in a seahorse plate at 6 × 103 cells per well 24 h before treatments were started. Cell were treated with Bafilomycin A1 (200 nM) (Thermofisher Scientific, Cat # SML1661) or Bafilomycin A1 plus acNPs (at concentrations from 30 µg/mL to 200 µg/mL) for 5 h. Cells were incubated under glucolipotoxicity (GLT, 150 µM BSA-palmitate plus 25 mM glucose) or GLT plus acNPs (30 µg/mL to 200 µg/mL) for 18 h. Respiration was assessed using a Seahorse bioanalyser in an assay media containing 10 mM glucose (Sigma Aldrich, Cat # G7021), 2 mM pyruvate (Sigma Aldrich, Cat # P76225) and 4 mM glutamine (Sigma Aldrich, Cat # G3126). Injections were as follows: oligomycin in port A (2 µM final concentration) (EMD Millipore, Cat # 495455), FCCP (in bafilomycin treated cells) (Enzo Life Sciences, Cat # BML-CM120-0010) or BAM 15 (in GLT treated cells) (Cayman Chemical Company, Cat # 17811) in port B (1.5 µM or 10 µM) and antimycin A in port C (2 µM) (Enzo Life Sciences, Cat # ALX-380-075-M010). Port injection mix were prepared in assay media but for BAM 15 that was prepared in 600 µM oleate, 100 µM glutamine and 1 mM pyruvate. Cells were fixed after the assay and stained with DAPI. Cell counts were assessed using the Operetta system.
5
Lysosomal Trafficking of Endogenous NPY
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To test for trafficking of endogenous NPY to the lysosome, two different methods for blocking lysosomal degradation were used. After 3 days of knockdown, media was replaced with media containing either Bafilomycin A1 (Alfa Aesar) at 200 nM concentration for 18 hours, or media containing 10 µM E64 and 50 µM Leupeptin (both Sigma Aldrich) for 24 hours. After this time, cells were lysed and the levels of endogenous NPY quantified by western blot. For co-localisation analyses of NPY with Lamp1 in the presence of Bafilomycin A1 the experiment was carried out as above with cells on coverslips and 18 hours incubation in BafA1.
6
Neuropsychopharmacological Agents Protocol
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The following drugs were used: amitriptyline hydrochloride (Sigma, A8404), bafilomycin A1 (Alfa Aeser, J61835), dexamethasone (Sigma, D1756), fluoxetine hydrochloride (Sigma, F132), haloperidol (Sigma, H1512), ketamine hydrochloride (Sigma, K2753), paroxetine hydrochloride hemihydrate (Sigma, P9623), and scopolamine hydrobromide (Sigma, S1875).
7
Drug Treatments for Cellular Processes
8
Hippocampal Neuron Culture and Lentiviral Transduction
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Fetal WT hippocampal tissue (gestation E15) were dissected and gently triturated with a fire-polished Pasteur pipette, and the resulting pool of dissociated cells were plated in 35-mm dishes precoated with polyethyleneimine (1 mg/ml) (MP Biomedicals NC-N19544450) containing 0.5 ml basal growth medium, which was supplemented with 5% horse serum and 0.5% fetal calf serum and replaced with fresh medium every three days. DIV 14 or when the density of cells reached 3×104 cells, neurons were infected with 3 μl lentiviruses for 24 hrs and then treated with 10 μM Nilotinib (AMN-107, NC0604306, SELLECK CHEMICAL LLC) or 1 μL DMSO for 24 hrs or 100 nM Bafilomycin-A1(AC32812-0001, Acros Organics) or 20 μM MG132 (NC9819784, Cayman Chemicals) for 6 hrs.
9
Synthesis of Functionalized Polyethylene Glycol
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All manipulations were carried out in a nitrogen-filled glove box. mPEG-OH (5000 g/mol, Aladdin, https://www.aladdin-e.com/ ) was dried in vacuum at 60 °C for 24 h prior to use. Allyl glycidyl ether (AGE, 98%, Aladdin, https://www.aladdin-e.com/ ) was distilled after being dried over CaH2. Phthalic anhydride (PA, 99%, Aladdin, https://www.aladdin-e.com/ ) was purified by subliming several times under reduced pressure. Triethyl borane (Et3B in THF, 1.0 mol/L, Aladdin, https://www.aladdin-e.com/ ), t-BuP1 (98%, Aldrich, https://www.sigmaaldrich.cn/CN/zh ), 2,2′-Azobis(2-methylpropionitrile) (AIBN, Aladdin, https://www.aladdin-e.com/ ) were all used directly without purification. N-(2-(3′,6′-bis(diethylamino)-3-oxo-4a′,9a′-dihydrospiro[isoindoline-1,9′-xanth-en]-2-yl)ethyl)-2 mercap-toacetamide (RhB-SH) was synthesized according to the literature procedures [57 (link)], Bafilomycin A1 (ACROS, https://www.acros.com/ ).
10
Autophagy Regulation in TGF-β1 Signaling
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The V-ATPase inhibitor Bafilomycin A1 (ACROS Organics; A0360555) was used at a concentration of 100 nM. TGFβ1 (R&D Systems; 240B0020) was used at a concentration of 10 ng/ml. The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.
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