Bafilomycin

Manufactured by InvivoGen

Sourced in United States, France

Bafilomycin is a macrolide compound that inhibits vacuolar-type H+-ATPase (V-ATPase). It is commonly used as a research tool to study cellular processes involving V-ATPase, such as vesicle trafficking and acidification of organelles.

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Lab products found in correlation

Pam3csk4, by InvivoGen (4 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Bx795, by InvivoGen (2 mentions) Tnf α, by Thermo Fisher Scientific (2 mentions) Itacitinib, by MedChemExpress (2 mentions) Ru 521, by InvivoGen (2 mentions) Odn20959, by Miltenyi Biotec (2 mentions) Mrt67037, by InvivoGen (2 mentions) H 151, by InvivoGen (2 mentions) Ruxolitinib, by MedChemExpress (2 mentions) Rapamycin, by InvivoGen (2 mentions) L glutamine penicillin streptomycin solution, by Merck Group (1 mentions) Rpmi 1640 medium, by Harvard Bioscience (1 mentions) Fetal bovine serum (fbs), by PAN Biotech (1 mentions) Epirubicin, by Merck Group (1 mentions) Phorbol 12 myristate 13 acetate pma, by Abcam (1 mentions) Lps from e coli k12, by InvivoGen (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Macs isolation kit, by Miltenyi Biotec (1 mentions) Zombie aqua reagent, by BioLegend (1 mentions)

Spelling variants of this product

Bafilomycin A1 (57 citations) Bafilomycin A (4 citations) Bafilomycin A1 (tlrl-baf1) (4 citations) Bafilomycin A1 (BafA1) (3 citations) Bafilomycin A1 (Cat. tlrl-baf1) (2 citations)

11 protocols using bafilomycin

Epirubicin and Inflammasome Activation in THP-1 Cells

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Cells were cultivated in RPMI-1640 medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS; Pan Biotech, Aidenbach, Germany) and 1% L-glutamine-penicillin-streptomycin solution (GPS; Sigma-Aldrich, St. Louis, Missouri, USA). Prior to further stimulation, THP-1 were differentiated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Abcam, Cambridge, UK) for 18 h.
Cells were treated with freshly prepared, sterile-filtered epirubicin (Sigma Aldrich) at concentrations between 0.01 to 5 µg/mL for 24 h, unless stated otherwise. After epirubicin treatment, PM and THP-1 were stimulated for 3 h with LPS from E.coli K12 (InvivoGen, San Diego, CA, USA) at concentrations of 10 or 100 ng/mL, respectively, followed by treatment with freshly prepared 1 mM ATP (InvivoGen, San Diego, CA, USA) for 1 h. For the inhibition of autophagy, 300 nM bafilomycin (Invivogen, San Diego, CA, USA) or 10 mM 3-methyl adenine (3-MA; Invivogen, San Diego, CA, USA) were added to the cells simultaneously with LPS, and maintained until the end of the experiment. For TLR2 ligation, cells were incubated either with 10⁸ cells/mL of heat-killed listeria monocytogenes (HKLM; Invivogen, San Diego, CA, USA) or 100 ng/mL Pam3CSK4 (Invivogen, San Diego, CA, USA) for 4 h.

Köse-Vogel N., Stengel S., Gardey E., Kirchberger-Tolstik T., Reuken P.A., Stallmach A, & Bruns T. (2019). Transcriptional Suppression of the NLRP3 Inflammasome and Cytokine Release in Primary Macrophages by Low-Dose Anthracyclines. Cells, 9(1), 79.

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Immune Response Modulation Protocol

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PAM3CSK4 was purchased from Invivogen and was reconstituted in endotoxin‐free H₂O. IFN‐α and IFN‐β were purchased from PBL Interferon Source. B18R was purchased from R&D Systems. TNF‐α was purchased from PeproTech. PMA (Sigma), TLR3 inhibitor (Calbiochem), TLR7/8 inhibitor ODN20959 (Miltenyi), A151 (Invivogen), ruxolitinib (MedChemExpress), itacitinib (MedChemExpress), bafilomycin (Invivogen), BX795 (Invivogen), MRT67037 (Invivogen), H‐151 (Invivogen), G150 (Selleckchem), and RU.521 (Invivogen) were reconstituted in DMSO or sterile deionized water.

Dickey L.L., Martins L.J., Planelles V, & Hanley T.M. (2022). HIV‐1‐induced type I IFNs promote viral latency in macrophages. Journal of Leukocyte Biology, 112(5), 1343-1356.

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Treg Cell Dynamics with miR-181a/b-1 Knockout

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CD4⁺CD25⁺ Treg cells were enriched from spleens and LNs of miR-181a/b-1^+/− or miR-181a/b-1^−/− mice using a MACS isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were incubated in the absence or presence of 50 nM bafilomycin (InvivoGen, San Diego, CA, USA) and collected after 30, 60, 120, and 180 min. Samples were stained with αCD4, αCD25, and αhCD2 (Foxp3) antibodies and intracellularly for CTLA-4. For intracellular stainings, the Foxp3/transcription factor staining buffer set (eBioscience) was used according to the manufacturer’s protocol. Samples were acquired on LSRII (BD Biosciences). Data were analyzed with FlowJo software, v.9.4.9 (Tree Star). For analysis, dead cells and debris were excluded via staining with Zombie Aqua reagent (BioLegend) prior to fixation and permabilization of cells.

Łyszkiewicz M., Winter S.J., Witzlau K., Föhse L., Brownlie R., Puchałka J., Verheyden N.A., Kunze-Schumacher H., Imelmann E., Blume J., Raha S., Sekiya T., Yoshimura A., Frueh J.T., Ullrich E., Huehn J., Weiss S., Gutierrez M.G., Prinz I., Zamoyska R., Ziętara N, & Krueger A. (2019). miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity. PLoS Biology, 17(3), e2006716.

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Standardization of TLR Agonists and Antagonists

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The stimulants LPS, R848, ssRNA40, and PAM₃CSK₄ and the antagonists bafilomycin, polymyxin B (PMB), dynasore, and CLI-095 were purchased from Invivogen (Toulouse, France). The concentration of above stimulants and antagonists were standardised as LPS (10 ng/mL), R848 (2 µg/mL), ssRNA40 (5 µg/mL), PAM₃CSK₄ (2 µg/mL), bafilomycin (1 µM), PMB (10 µg/mL), Dynasore (50 µM), and CLI-095 (3 µM). Mycobacterial RNA was extracted from gamma-irradiated Mycobacterium tuberculosis H37Rv (BEI Resources, NR-14819) with InnuPrep RNA Mini Kit (Analytik Jena, Germany). Purity was confirmed by Scandrop analysis (Analytik Jena). A 260 nm/280 nm extinction quotient of 1.9-2.0 was considered pure. For transfection, if not otherwise specified, LyoVec (Invivogen) was used in a 3 µg/100 µL dilution according to protocol.

Thada S., Horvath G.L., Müller M.M., Dittrich N., Conrad M.L., Sur S., Hussain A., Pelka K., Gaddam S.L., Latz E., Slevogt H., Schumann R.R, & Burkert S. (2021). Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis. International Journal of Molecular Sciences, 22(4), 1560.

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Reconstitution and Preparation of Immune Reagents

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PAM3CSK4 was purchased from Invivogen and was reconstituted in endotoxin-free H₂O. IFN-α and IFN-β were purchased from PBL Interferon Source. B18R was purchased from R&D Systems. TNF-α was purchased from PeproTech. PMA (Sigma), TLR3 inhibitor (Calbiochem), TLR7/8 inhibitor ODN20959 (Miltenyi), A151 (Invivogen), ruxolitinib (MedChemExpress), itacitinib (MedChemExpress), bafilomycin (Invivogen), BX795 (Invivogen), MRT67037 (Invivogen), H-151 (Invivogen), G150 (Selleckchem), and RU.521 (Invivogen) were reconstituted in DMSO or sterile deionized water.

Dickey L.L., Martins L.J., Planelles V, & Hanley T.M. (2022). HIV‐1‐induced type I IFNs promote viral latency in macrophages. Journal of Leukocyte Biology, 112(5), 1343-1356.

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JAK2 Mutant Cell Line Experiments

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The human cell lines HEL (expressing JAK2^V617F), SET-2 (expressing both JAK2^V617F and JAK2^WT) and HL-60 (expressing JAK2^WT) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were grown in RPMI 1640 medium with Glutamax (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco).
Ruxolitinib and LB-100 were purchased from Selleck Chemicals (Houston, TX, USA). These drugs were used at a concentration of 1 µM and 2.5 µM, respectively, apart from in clonogenic assays where they were used at 250 nM and 1 µM. Bafilomycin (25 nM) was purchased from Invivogen (Toulouse, France). Chloroquine and Lys05 were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Chloroquine was used at 20 µM in cell lines, 10 µM in primary cultures and at 1 µM for clonogenic assays. Lys05 was used in vitro at a concentration of 5 µM, except for in clonogenic assays, in which it was used at 1 µM. SAR405 (3 µM) and Okadaic acid (OA, 5 nM) were purchased from APExBIO (Houston, TX, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively.

Courdy C., Platteeuw L., Ducau C., De Araujo I., Boet E., Sahal A., Saland E., Edmond V., Tavitian S., Bertoli S., Cougoul P., Granat F., Poillet L., Marty C., Plo I., Sarry J.E., Manenti S., Mansat-De Mas V, & Joffre C. (2023). Targeting PP2A-dependent autophagy enhances sensitivity to ruxolitinib in JAK2V617F myeloproliferative neoplasms. Blood Cancer Journal, 13(1), 106.

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Mtb Infection Assay with Macrophages

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Where indicated, macrophages were pre-treated with R10 media containing 20 μM E64d (Cayman), 0.5 μM MG-132 (Cayman), or 10 nM bafilomycin (InvivoGen), or DMSO alone (or other concentration of drug where indicated). The culture media was supplemented with an equal concentration of drug during infection with Mtb, and after washing off extracellular Mtb. Where indicated, the culture media was supplemented with 10 ng/mL of IFN-β after infection with Mtb.

Leddy O., White F.M, & Bryson B.D. (2023). Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I. eLife, 12, e84070.

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Listeria Infection of Macrophages

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The recombinant Listeria strains have been previously described [24 (link)]. For in vitro infections, they were grown to an OD₆₀₀ of 0.6–1.0 in BHI media (Sigma Aldrich) with 10 ug/ml chloramphenicol (Sigma Aldrich) at 30°C. Macrophages were infected with the Listeria strains using a MOI 50, for 45 minutes. Extracellular bacteria were eliminated by adding 60 ug/ml gentamicin (Sigma Aldrich) for 20 minutes. Bacterial burden was assessed by lysing the infected macrophages with 1% TritonX-100 in PBS, and plating serial dilutions of the lysate on BHI agarose supplemented with 10ug/ml chloramphenicol (Sigma Aldrich, St. Louis, MO). Recombinant listeriolysin (Prospec, East Brunswick, NJ) was added in some experiments at 2 ug/ml for 30 minutes, and any excess was washed away. Bafilomycin (InvivoGen, San Diego, CA) was added in some experiments at 5 uM for 30 minute, before being washed away.

Yang J.D., Mott D., Sutiwisesak R., Lu Y.J., Raso F., Stowell B., Babunovic G.H., Lee J., Carpenter S.M., Way S.S., Fortune S.M, & Behar S.M. (2018). Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages. PLoS Pathogens, 14(5), e1007060.

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Autophagy regulation by GAS6 and ADAM17

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Recombinant (r) GAS6 was purchased from R&D systems (Minneapolis, MN). The autophagy inducer, rapamycin, and the autophagy inhibitor, bafilomycin, were obtained from Invivogen (San Diego, CA). The ADAM17 inhibitor, TAPI-0 was purchased from Sigma Aldrich (St. Louis, MO).

Mulla M.J., Weel I.C., Potter J.A., Gysler S.M., Salmon J.E., Peraçoli M.T., Rothlin C.V., Chamley L.W, & Abrahams V.M. (2018). Antiphospholipid Antibodies Inhibit Trophoblast Toll-like Receptor and Inflammasome Negative Regulators. Arthritis & rheumatology (Hoboken, N.J.), 70(6), 891-902.

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Inducing Mitochondrial Stress and Autophagy

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iHF and MEF were cultivated at 37 °C and 5% CO₂ in DMEM—with (iHF) or without (MEF) GlutaMAX—and supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin (both, Life Technologies, Carlsbad, CA, USA). Mitochondrial stress was induced by cultivating cells in growth medium containing 50 µM of carbonyl cyanide m-chlorophenyl hydrazone (CCCP, Biomol, Hamburg, Germany) for 6 h. Autophagy was induced by cultivating cells in growth medium containing 400 nM of rapamycin (Merck Millipore, Burlington, MA, USA) for 6 h. To induce autophagy and block the fusion of autophagosomes and lysosomes, cells were cultivated in growth medium containing 400 nM of rapamycin for 4 h, followed by a 2 h cultivation in fresh growth medium containing 400 nM of rapamycin and 50 nM of bafilomycin (Invivogen, San Diego, CA, USA). Controls were cultivated in growth medium containing dimethyl sulfide (DMSO, Roth, Germany), which was used as a solvent for rapamycin and bafilomycin for equivalent time periods.

Harmuth T., Weber J.J., Zimmer A.J., Sowa A.S., Schmidt J., Fitzgerald J.C., Schöls L., Riess O, & Hübener-Schmid J. (2022). Mitochondrial Dysfunction in Spinocerebellar Ataxia Type 3 Is Linked to VDAC1 Deubiquitination. International Journal of Molecular Sciences, 23(11), 5933.

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