DC or HepG2 cells were pretreated with inhibitors in serum-free media for 30 min at 37 °C. Without washing, fluorescently-labeled protein was added at 10 μg/ml for 1 hr at 37 °C. After washing, MFI of internalized protein was quantitated by flow cytometry. Percent inhibition was calculated as: [(MFI of untreated cells) – (MFI of treated cells)]/(MFI of untreated cells) × 100 %. The following blocking antibodies were used: CD206 (clone 15–2; BioLegend), DC-SIGN (clone 120507), SR-A1 (clone 351620), LOX-1 (clone 331212; all from R&D Systems), CD36 (clone 185-1G2; NeoMarkers), and SR-B1 (rabbit polyclonal; Novus Biologicals). Dimethylamiloride (DMA; 100 μM), mannan (300 μg/ml), and polyinosinic acid (Poly I; 50 μg/ml) were purchased from Sigma.
Dimethylamiloride dma
Manufactured by Merck Group
Sourced in Macao
Dimethylamiloride (DMA) is a laboratory reagent used for research purposes. It is a sodium-hydrogen exchanger inhibitor that can be used to study cellular processes involving ion transport. The core function of DMA is to inhibit the sodium-hydrogen exchange activity in cells, which can be useful for various experimental applications. No further interpretation or extrapolation on the intended use of this product is provided.
Automatically generated - may contain errors
Lab products found in correlation
Cd206 clone 15 2, by BioLegend (1 mentions)
Sr b1, by Novus Biologicals (1 mentions)
Polyinosinic acid poly 1, by Merck Group (1 mentions)
Ethylisopropyl amiloride eipa, by Merck Group (1 mentions)
Y 27632, by Cayman Chemical (1 mentions)
Nrk 49f, by Merck Group (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Tgf β1, by Merck Group (1 mentions)
Nrk 49f, by American Type Culture Collection (1 mentions)
Nystatin, by Merck Group (1 mentions)
Colchicine, by Merck Group (1 mentions)
Chlorpromazine hydrochloride cpz, by Merck Group (1 mentions)
Exosome isolation reagent, by RiboBio (1 mentions)
6 protocols using dimethylamiloride dma
1
Receptor-mediated Protein Internalization Assay
2
Vasoconstriction Regulation Mechanisms
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ET‐1 (EMD Millipore) was used at 10−8 mol/L. The vehicle for ET‐1 was diH2O. 10 μmol/L Y‐27632 (Cayman Chemical, Ann Arbor, MI) was used to inhibit ROCK. The vehicle for Y‐27632 was diH2O. 10 μmol/L ethyl‐isopropyl amiloride (EIPA; Sigma) or 1 μmol/L dimethyl amiloride (DMA; Sigma) was used to inhibit NHE. The vehicle for both EIPA and DMA was dimethyl sulfoxide (DMSO).
3
Exosomal Signaling in Kidney Fibrosis
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Human proximal tubular epithelial cells (HK-2) and normal rat kidney interstitial fibroblast cells (NRK-49F) were obtained from the American Type Culture Collection (Manassas, VA). After serum starvation overnight, HK-2 cells were treated with 2 ng/ml recombinant human TGF-β1 (R & D Systems, Minneapolis, MN) for 6 h and then TGF-β1 was removed, followed by incubating in serum-free medium for 48 h. In some experiments, HK-2 cells were pretreated with 100 µg/ml dimethyl amiloride (DMA) (Sigma) or transfected with TNFAIP8 siRNA using Lipofectiamine 2000 (Life Technologies, Grand Island, NY). The conditioned media were collected and subjected to exosomes isolation. Following serum starvation for 16 h, NRK-49F cells were treated with HK-2 cell conditioned media or exosomes (30 µg) isolated from HK-2 cell conditioned media. For some experiments, NRK-49F cells were pretreated with cisplatin 20 µM (Sigma, St. Louis, MO) to induce cell apoptosis, followed by incubation with 10 µM p53 inhibitor Pifithrin-α (Selleck Chemicals) or MG132 (Selleck Chemicals), respectively. Cells after different treatments were collected and subjected to various analyses.
4
ELM Secretion Inhibition in Lung Cancer
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The human non-small cell lung cancer cell line H460 and mouse Lewis lung cancer cell line LLC1 were obtained from ATCC. Cells were grown in DMEM medium with glutamax-I supplemented with 10% (v/v) FBS and Pen/Strep in an atmosphere of 95% air and 5% CO2 at 37°C. H460 cells were transfected with the plasmid encoding hCD63-GFP, pCT-CD63-GFP (SBI, USA). Stable H460-hCD63-GFP cells were selected and maintained in puromycin-containing medium at 1 µg/ml. Dimethyl amiloride (DMA) (Sigma, MO) was used to inhibit ELM secretion in H460-hCD63-GFP cells [40] (link). Cells were treated with 1 µmol or 10 µmol DMA for 48 h, and the amount of ELMs secreted into medium in the presence of DMA was determined and compared to untreated controls using an acetylcholinesterase activity assay. The trypan blue exclusion assay was used to determine the cytotoxity of DMA using a Beckman Coulter Vi-Cell (Beckman Coulter Inc., CA).
5
Transcytosis of ZIKV across Polarized Cell Barrier
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Cells were seeded to 24-well PET cell culture inserts, 0.4-μm pore size for 7 days, in a two-chambered system. In this system, barrier cells form a polarized monolayer with tight junctions between cells, allowing access to both apical and basolateral domains. Cells were incubated for 30 min with various transcytosis inhibitors in different concentration, including 10 μM of nystatin (Sigma), 10 μM of chlorpromazine hydrochloride (CPZ, Sigma), 20 μM of dimethyl amiloride (DMA, Sigma), and 10 μM of colchicine (Sigma). Drugs were administered to the cells in both apical and basolateral chambers at identical concentrations. Thereafter, 100 μl of Atto647-ZIKV (2 × 106 copies ml–1 of virus) was added into the upper chamber. After 4 h incubation with cells, all of the basolateral supernatant was collected. Percent transcytosis was determined by measuring the fluorescence intensity of Atto647N through using a fluorescent plate reader.
6
Exosome Isolation and Inhibition
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Exosomes were separated from SCLC cell cultures using the Exosome Isolation Reagent (Ribo) following the manufacturer’s instructions. First, the cell culture was collected and centrifuged (2000 × g, 30 min) to remove residual cells. The supernatant was transferred to a new tube to which 1/3 volume of isolation reagent was added and mixed. The mixture was refrigerated overnight at 4 °C and then centrifuged (1500 × g, 30 min). Sediments were collected for further analysis. The size and distribution of exosomes were verified using transmission electron microscopy (TEM; JEOL). For the inhibition of exosome release, SCLC cells were typically incubated with 15 nM of the pharmacological inhibitor, Dimethyl amiloride (DMA; Sigma) for 24 h.
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