Bafilomycin a1

Manufactured by Cell Signaling Technology

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Bafilomycin A1 is a macrolide antibiotic that inhibits vacuolar-type H+-ATPase (V-ATPase). V-ATPases are responsible for acidifying various intracellular compartments, such as lysosomes, endosomes, and secretory vesicles. Bafilomycin A1 acts as a specific and potent inhibitor of V-ATPase, preventing the acidification of these cellular compartments.

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Bafilomycin A1 (Baf A1; Bafilomycin

22 protocols using bafilomycin a1

Isolation and Differentiation of Human Microglia

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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of HIV seronegative donors using density gradient centrifugation over Ficoll–Paque Plus (GE Healthcare Cat# 114402). Primary human monocytes were isolated from PBMCs using monocyte isolation kit II, human (Miltenyi Biotech Cat# 130–091-103). Human microglia cells (HMG) were differentiated from primary human monocytes using methods as described previously (Etemad, Zamin, Ruitenberg, & Filgueira, 2012 (link); Rawat & Spector, 2017 (link)). Briefly, monocytes were seeded at 2.5×10⁵ cells/well in a 48-well plate in serum free RPMI Glutamax medium (Gibco Cat# 61870036) supplemented with MCSF (10ng/ml; Peprotech Cat# 300–25), MCP1 (100ng/ml; Peprotech Cat# 300–04), GMCSF (10ng/ml; Peprotech Cat# 300–03) and NGF-β (10ng/ml; R&D Systems Cat# 256-GF) with media changed every 3 days.
For autophagy induction in HMG cells, culture medium was supplemented with trehalose (Sigma, Cat# T0167) at 100mM concentration. The lysosomal degradation inhibitor bafilomycin A1 (Cell Signaling Cat# 54645) was prepared in dimethyl sulfoxide (DMSO) and added to the medium at a concentration of 10nM to measure the autophagic flux. DMSO was added to the medium of untreated cells as vehicle control.

Rawat P., Diedrich C.T, & Spector S.A. (2018). Human Immunodeficiency Virus Type-1 single-stranded RNA activates the NLRP3 inflammasome and impairs autophagic clearance of damaged mitochondria in human microglia. Glia, 67(5), 802-824.

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Lipid Standards and Cell Line Protocols

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Synthetic lipid standards, chemicals, and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA), Rathburn Chemicals (Walkerburn, Scotland), Avanti Polar Lipids (Alabaster, AL, USA), and Larodan AB (Solna, Sweden), unless otherwise specified. Cell lines used were human female cervix cancer cell line (HeLa, a kind gift from Prof. J. Bartek, Danish Cancer Institute, Copenhagen, Denmark) and human female osteosarcoma cell line (U2OS, ATCC HTB-96, VA, USA), and human female ductal breast carcinoma cell line (a highly tumor necrosis factor-sensitive subclone of MCF-7 cells (Jaattela, 1995 (link)), hereafter called MCF7). Cell culturing media and supplements were purchased from Life Technologies (Carlsbad, CA, USA).
Siramesine (kind gift from Lundbeck A/S, Copenhagen, Denmark), ebastine (Cayman Chemical, #15372), loratadine (Sigma-Aldrich, L9664), L-leucyl-L-leucine methyl ester (Santa Cruz Biotechnology, #SC-285992), and bafilomycin A1 (Cell Signaling Technology, #54645) were all dissolved in DMSO. Lysoglycerophatidylcholine (LPC) 17:0 (Avanti Polar Lipids, USA, 855676C) was dissolved in chloroform. To supply it into cell culture media, they were vacuum centrifuged for 20 min and then dissolved in FCS by shaking at 2000 rpm and 4°C for 16 h. For a list of chemicals, reagents and technical equipment, and their suppliers, see Supplemental Tables S4 and S5.

Nielsen I.Ø., Clemmensen K.K., Fogde D.L., Dietrich T.N., Giacobini J.D., Bilgin M., Jäättelä M, & Maeda K. (2024). Cationic amphiphilic drugs induce accumulation of cytolytic lysoglycerophospholipids in the lysosomes of cancer cells and block their recycling into common membrane glycerophospholipids. Molecular Biology of the Cell, 35(3), ar25.

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Immunofluorescence Staining of Autophagy Markers

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Anti-LC3A/B rabbit polyclonal antibody was purchased from Cell Signaling Technology. Anti-p62 mouse monoclonal antibody and Alexa®Fluor 647-labeled Anti-LAMP1 mouse monoclonal antibody were purchased from Santa Cruz Biotechnology. Anti-ATPB mouse monoclonal antibody was purchased from ABCAM. Anti-LAMP3 (CD63) mouse monoclonal antibody was purchased from BD Pharmingen. Alexa®Fluor 594 donkey-anti-rabbit, Alexa®Fluor 594 goat-anti-mouse secondary antibodies, anti-GFP rabbit monoclonal antibody, and goat-anti-mouse secondary antibody HRP were purchased from Life Technologies. Anti-ATG5 mouse monoclonal antibody was purchased from MBL. Anti-GAPDH mouse monoclonal antibody was purchased from Sigma Aldrich. Pooled Ig from HIV-1-infected patients (HIV-Ig) was obtained from the NIH AIDS Research and Reference Reagent Program. Rapamycin and leupeptin were purchased from Sigma Aldrich. Bafilomycin A1 was purchased from Cell Signaling Technology. DAPI was purchased from Life Technologies.

Qu N., Ma Z., Zhang M., Rushdi M.N., Krueger C.J, & Chen A.K. (2017). Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation. Protein & Cell, 9(7), 640-651.

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Senescence and Oxidative Stress Assay

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Cells positive for reactive oxygen species and senescence-associated β-galactosidase were quantified using flow cytometry. Senescence-associated β-galactosidase was evaluated using the C₁₂FDG substrate which becomes fluorescent when hydrolyzed by SA-β-galactosidase. ADSCs were tested at passages 4, 6, and 8 after being plated in 12-well plates (Genesee, catalog # 25106) in triplicate at a density of 40 000 cells/well. The cells were treated with 100 nM of bafilomycin A1 (Cell Signaling, catalog # 54645) for 1 h at 37°C and 5% CO₂. Next, 33µM C₁₂FDG (Thermo Fisher, catalog # D2893) was added and incubated for 2 h. After 1.5 h the CellROX reagent (Thermo Fisher, catalog # C10422) was also added for the remaining 30 min. The cells were then washed with PBS and collected from the plate using the TrypLE reagent. Using the Guava EasyCyte flow cytometer, cells positive for C₁₂FDG and reactive oxygen species were quantified using unstained cells to set gate thresholds for fluorescence.

Mullen M., Nelson A.L., Goff A., Billings J., Kloser H., Huard C., Mitchell J., Hambright W.S., Ravuri S, & Huard J. (2023). Fisetin Attenuates Cellular Senescence Accumulation During Culture Expansion of Human Adipose-Derived Stem Cells. Stem Cells, 41(7), 698-710.

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Cell Viability Assay with Inhibitors

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Cell growth over time was measured using the PrestoBlue Cell Viability Reagent (Invitrogen). Briefly, 67NR, 5637, SW480 or MDA-468 cells were seeded in 96-well plates (2,500 cells/well) and exposed to ATX-101 (25 amino acid cell-penetrating APIM-containing peptide [21 (link)], Ac-MD-RWLVK-W-KKKRK-I-RRRRRRRRRRR, Invitrogen, Sweden) (4-12 μM), ATX-A (Ac-MD-RALVK-W-KKKRK-I-RRRRRRRRRRR, Invitrogen, Sweden) (6 μM), AEE788 (EGFR/HER2/VEGFR inhibitor, Selleck Chem) (0.5-1 μM), Bafilomycin A1 (10 nM) Cell Signaling, #54645, or cMet inhibitor (PHA-665752, Sigma, 2 μM) until harvest at day three. Data displayed are percentage viability relative to untreated control in one representative experiment out of three biological replicas demonstrating the same trend.

Søgaard C.K., Nepal A., Petrovic V., Sharma A., Liabakk N.B., Steigedal T.S, & Otterlei M. (2019). Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy. Oncotarget, 10(68), 7185-7197.

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EVs Uptake by HS-5 Cells

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EVs were labeled with 2.5 mM PKH67 green fluorescent dye using PKH67 Green Fluorescent Cell Linker Midi Kit (Sigma-Aldrich) in 400 ml diluent C for 5 min, blocked with 1% bovine serum albumin (BSA) for 1 min, and then washed with PBS at 120,000 g for 2 h at 48℃. Subsequently, PKH67-labeled EVs were resuspended in PBS and stored at -80℃. A total of 1.5 × 10⁵ HS-5 cells were incubated with 4 × 10⁶ PKH67-labeled EVs in 96-well plates for 18 h, or treated with V-ATPase inhibitor [Bafilomycin A1, # 54,645, Cell Signaling Technologies (CST), Beverly, MA, USA]. After undergoing staining with DAPI, the uptake of EVs was observed under a confocal microscope (LSM780, Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA).

Hu C.Y., Chen J., Qin X.H., You P., Ma J., Zhang J., Zhang H, & Xu J.D. (2021). Long non‐coding RNA NORAD promotes the prostate cancer cell extracellular vesicle release via microRNA-541-3p-regulated PKM2 to induce bone metastasis of prostate cancer. Journal of Experimental & Clinical Cancer Research : CR, 40, 98.

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Culturing and Treating Human Cell Lines

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Human embryonic kidney 293T cells and human hepatocyte carcinoma HepG₂ cells were purchased from American Type Culture Collection (ATCC, Manassas, VA), the CRBN knock-out HEK293T cell line was a kind gift from Dr. Michael Erb (Scripps Research, La Jolla, USA), and the ATG 2A/2B ^−/− cell line was a kind gift from Dr. Masaaki Komatsu (Juntendo University, Tokyo, Japan). The cell lines were cultured in high-glucose (4.5 g/L) Dulbecco’s Modified Eagle’s Medium (HyClone, Marlborough, MA). The media was supplemented with 10% fetal bovine serum (VWR, Radnor, PA), 100 units/mL of penicillin, and 100 mg/mL of streptomycin (Thermo Fisher Scientific, Waltham, MA). The cells were cultured at 37 ℃ in a 5% CO₂ incubator. Cycloheximide (CHX, #C1988), MG132 (#474790), VER155008 (SML0271) were purchased from Sigma-Aldrich (St. Louis, MO), Bortezomib (BTZ, #NC0175953) was purchased from LC laboratory (Woburn, MA), bafilomycin A1 (BafA1, #54645S) was purchased from Cell Signaling Technology (Danvers, MA), thapsigargin (Tg, #1138) was purchased from R & D SYSTEMS Inc. (Minneapolis, MN), and E3 ligase recruiter building blocks thalidomide - linker 2 (#6468/25) were purchased from Tocris Bioscience (Minneapolis, MN) and VHL Ligand 1(#21591) was purchased from Caymen Chemical (Ann Arbor, MI). The detailed synthetic procedure is described in the Supporting Information.

Mingot J.M., Espeso E.A., Díez E, & Peñalva M.Á. (2001). Ambient pH Signaling Regulates Nuclear Localization of the Aspergillus nidulans PacC Transcription Factor. Molecular and Cellular Biology, 21(5), 1688-1699.

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MTT Assay for Cell Viability Analysis

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MTT was obtained from Abcam (MTT Assay Kit, Abcam ab211091, Cambridge, UK). A549 and H460 cells were seeded at a density of 0.5–1 × 10⁴ cells/mL in 96 well plates. The plates were then treated with concentrations of 0.2% WPSC for 24, 48 and 72 h. 200 µM hydrogen peroxide treatment was used as a positive control for 25 min. Cells were treated with 100 nM Bafilomycin A1 (Cell Signaling, 54645, Danvers, MA, USA), where indicated for 1 h prior to WPSC treatment. 20 μL of MTT solution (5 mg/mL) was added to each well and the cells were cultured for another 2 h. The treatment medium was then discarded and 50 µL of serum-free media and 50 µL of MTT Reagent were added together into each well and the plate was incubated at 37 °C for 3 h. Following incubation, 150 µL of MTT solvent was added to each well. The plate was covered in foil and agitated on an orbital shaker for 15 min then read at 590 nm using a microplate reader (BioTek Epoch 2, Winooski, VT, USA). Cell proliferation rates were calculated by comparing with the control cells.

Zaarour R.F., Sharda M., Azakir B., Hassan Venkatesh G., Abou Khouzam R., Rifath A., Nizami Z.N., Abdullah F., Mohammad F., Karaali H., Nawafleh H., Elsayed Y, & Chouaib S. (2022). Genomic Analysis of Waterpipe Smoke-Induced Lung Tumor Autophagy and Plasticity. International Journal of Molecular Sciences, 23(12), 6848.

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Titanium Modulates Autophagy in Osteoblasts

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The effect of Ti ions on autophagy in osteoblastic cells was evaluated. Cells were treated with TiCl₄ (cat. no. 7550-45-0; Shanghai Aladdin Bio-Chem Technology Co., Ltd.) at different concentrations (0, 10, 25 and 50 µM) at 37°C for 24 h. The cells were also treated with various concentrations (0, 10, 25 and 50 µM) of TiCl₄ for 24 h with or without lysosomal inhibitor bafilomycin A1 (Baf-A1; 20 nM; Cell Signaling Technology, Inc.) at 37°C for 24 h. The vehicle control used for each in vitro assay was 0.1% DMSO. The role of the sirtuin3 (SIRT3)/superoxide dismutase 2 (SOD2) pathway in osteoblasts was investigated.

Wang S., Yang J., Lin T., Huang S., Ma J, & Xu X. (2020). Excessive production of mitochondrion-derived reactive oxygen species induced by titanium ions leads to autophagic cell death of osteoblasts via the SIRT3/SOD2 pathway. Molecular Medicine Reports, 22(1), 257-264.

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Effect of Proteasome and Autophagy Inhibitors on MBNL2 Expression

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To test the effect of proteasome and autophagosome inhibitors on in the pINDUCER20_ FLAG-MBNL2(+E9) and pINDUCER20_FLAG-MBNL2(–E9) C2C12 cell lines, 150 000 cells per well were seeded in 6-well plates with DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (GenDepot), 1% Penicillin Streptomycin (Thermo Fisher Scientific) and 1 μg/ml Doxycycline hyclate (Sigma-Aldrich) at 37°C in 5% CO₂ to induce FLAG-MBNL2 expression. Forty-eight hours following cell seeding, the media was removed and fresh media including either 10 μM of the proteasomal inhibitors MG-132 (EMD Millipore), 1 μM of the proteasomal inhibitor Bortezomib (Fisher Scientific) or 1 μM of the autophagosome inhibitor bafilomycin A1 (Cell Signaling Technologies) was added to the cells. The cells were cultured for an additional 10 h and then harvested for western blot analyses.

Nitschke L., Hu R.C., Miller A.N., Lucas L, & Cooper T.A. (2023). Alternative splicing mediates the compensatory upregulation of MBNL2 upon MBNL1 loss-of-function. Nucleic Acids Research, 51(3), 1245-1259.

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