Dopamine hydrochloride (DA), epinephrine bitartrate salt (EP), norepinephrine bitartrate salt monohydrate (NEP), sodium monobasic phosphate (Na2HPO4), sodium dibasic phosphate (NaH2PO4), potassium chloride (KCl), potassium ferricyanide (III) (K3[Fe(CN)6]), potassium ferrocyanide (II) (K4[Fe(CN)6]), gold (III) chloride solution, and ammonium chloride (NH4Cl) were purchased from Sigma-Aldrich (Buchs, Switzerland). All solutions were prepared in phosphate buffer 0.1 M, KCl 0.1 M, pH 7.4 (PBS). High-purity deionized water (resistance: 18.2 MΩ cm at 25 °C; TOC < 10 µg L−1) obtained from Millipore (Molsheim, France) has been used throughout experiments.
Ammonium chloride nh4cl
Manufactured by Merck Group
Sourced in United States, Germany
Ammonium chloride (NH4Cl) is a white crystalline compound that is widely used in various laboratory applications. It is a salt composed of ammonium (NH4+) and chloride (Cl-) ions. Ammonium chloride is a common laboratory reagent and is known for its ability to serve as a pH buffer, conducting salt, and a source of ammonium ions.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Mg132, by Merck Group (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Dynasore, by Merck Group (3 mentions)
Wortmannin, by Merck Group (3 mentions)
Tris hcl, by Avantor (2 mentions)
Dithiothreitol (dtt), by GoldBio (2 mentions)
Cholesterol, by Avantor (2 mentions)
Sodium orthovanadate na3vo4, by Enzo Life Sciences (2 mentions)
Leupeptin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Acetophenone, by Merck Group (2 mentions)
Hexachloroplatinic acid hydrate, by Merck Group (2 mentions)
Isopropyl alcohol, by Merck Group (2 mentions)
Heptane, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Sodium citrate, by Merck Group (2 mentions)
Chloroquine, by Merck Group (2 mentions)
48 protocols using ammonium chloride nh4cl
1
Electrochemical Characterization of Neurotransmitters
2
Bovine Lactoferrin Purification and Characterization
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Lactoferrin from bovine milk (bLf) was obtained from Morinaga Milk Industries (Zama City, Japan). Endotoxin deprivation, purity checking, protein concentration, and iron saturation rate were assayed as previously described [19 (link),24 (link),25 (link)]. Detoxified bLf and ammonium chloride (NH4Cl, Sigma Chemical Co., St. Louis, MO, USA) were dissolved as stock solutions (0.25 mM and 400 mM, respectively) in pyrogen-free phosphate buffered saline (PBS, pH 7.4). Cytotoxicity was evaluated according to a previously reported method [19 (link)].
3
Purification of Cellular Organelles
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DSP and ammonium chloride (NH4Cl) were purchased from Sigma-Aldrich (St Louis, MO). BFA was purchased from BioLegend (San Diego, CA). Bortezomib was from Selleck Chemicals (Houston, TX). Carfilzomib was purchased from ApexBio Technology (Houston, TX).
4
Autophagy Pathway Inhibition Assay
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The following chemicals and drugs were used to inhibit the autophagy pathway: 20 mM ammonium chloride (NH4Cl) (Sigma) and 100 μM leupeptin (Enzo) to inhibit the lysosome activity; 1 μM (IF) or 10 μM (IB) vinblastine (Sigma) to inhibit the fusion between autophagosome and lysosome. 10 µM FK506 (Sigma) was used to inhibit the phosphatase calcineurin and 10 μM BAPTA-AM (Invitrogen) was used to chelate the cytosolic Ca2+. To inhibit protein translation and synthesis, 10 μg/ml cycloheximide (CHX) (Sigma) was used.
5
Isolation and Culture of Neonatal Rat Cardiomyocytes
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Isolation and culture of primary neonatal rat cardiomyocytes (NRCMs) were prepared from heart of 1- to 3-days old WT and MYL4p.E11K rats. In brief, the separated atriums and ventricles were digested in 1% collagenase II. The supernatant was stopped by culture medium (20% FBS, 1% penicillin and streptomycin in complete high glucose DMEM medium) and filtered by 100-μm cell strainer. Then collected supernatant supernatants was pre-plated for 60 min to remove fibroblasts and endothelial cells. The residual supernatant with cardiomyocytes was centrifuged at 300×g for 8 min and replanted in collagen-coated dishes at 5% CO2 and 37 °C for 24 h. Cells were treated with 30 mM ammonium chloride (NH4Cl) (Sigma, USA) for 12 h to inhibit endosome-lysosome system acidification.
6
Reconstitution of Membrane Transporters
7
Assessing Lysosomal Alkalinization via Flow Cytometry
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Lysosomal alkalinization was performed as reported elsewhere [32 (link)]. Briefly, OKF6/TERT-2 cells were incubated for 1 h with 200 nM bafilomycin A1 (Alexis Biochemicals, CA, USA), 10 µM chloroquine diphosphate salt (#C6628, Sigma-Aldrich), or 20 mM ammonium chloride (NH4Cl) (#254134, Sigma-Aldrich) to inhibit the downstream autophagic activity [5 (link)]. After washing out the autophagy inhibitors, the cells were incubated with 1 µM of acridine orange (#14338, Cayman chemical, Ann Arbor, MI, USA) for 20 min at 37 °C to evaluate the alkalinization of lysosomes (Supplementary Figure S1F–G ). The acridine orange is a permeable fluorescent cationic dichromatic probe that detects the pH-dependent changes in mammalian cells. This assay allows the detection of emission spectra of 525 nm (green) and 650 nm (red). The emission spectra correspond to the basification (green) and acidification (lysosomes in red) processes of the cell structures after the treatment with downstream autophagy inhibitors [33 (link)]. The samples were analyzed in the CytoFLEXTM V3-B3-R3 flow cytometer (#B53007, Beckman-Coulter Life Sciences, Brea, CA, USA) in PerCP detector (Ex-Max 488 nm/Em-Max 650–690 nm). The acquired data were analyzed using FlowJo v10.0.8 software (Tree Star Inc., Ashland, OR, USA).
8
Characterization of Lysosomal Dynamics
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Mouse monoclonal antibodies to LC3 (catalog #0231-100/LC3-5F10; Nanotools) were used for immunoblotting (1:300) experiments. Anti-LAMP antibodies (LAMP-2: catalog #ABL-93, 1/200; LAMP-1: catalog #H4A3 1:200) used for immunofluorescence were purchased from the Developmental Studies Hybridoma Bank. Anti–V-ATPase subunit V1D (D-4; catalog #SC-166218; Santa Cruz Biotechnology, Inc.) was used at 1:200 for immunofluorescence and 1:500 for immunoblotting. Anti–V-ATPase subunit V1B2 (catalog #AB73404; Abcam) was used at 1:200 for immunofluorescence and 1:500 for immunoblotting. LysoTracker red DND-99 (500 nM), Dextran conjugated with Fluorescein and Tetramethylrhodamine, 70,000 mol wt (2.5 mg/ml), BODIPY-FL–Pepstatin A, and DQ-BSA (10 µg/ml) were purchased from Thermo Fisher Scientific. Rabbit polyclonal anti-GAPDH (catalog #14C10; 1:5,000) and anti-Actin (catalog #13E5; 1:5,000) were purchased from Cell Signaling Technology. Ammonium chloride (NH4Cl), bafilomycin A1, KT5720, leupeptin, E64D, Pepstatin A, and MG132 were purchased from Sigma-Aldrich. Sp-8-cpt-cAMP was purchased from Biolog Life Science Institute. We applied secondary antibodies conjugated to Alexa Fluor 488 or 546 (IF 1:1,000). The MTT assay kit was bought from Roche, and the experiment was performed according to the manufacturer’s protocol.
9
Inhibition of Viral Replication and Assembly
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Ammonium chloride (NH4Cl, Sigma-Aldrich) at 20mM, Bafilomycin A1 (Baf A1; Cell Signaling) at 0.1µM, and chloroquine (CQ; Sigma-Aldrich) at 100µM were added to complete DMEM or DMEM without Methionine (Gibco). The media were then added to cells after viral adsorption (0 hpi) or at different time points of infection as specified. Guanidine hydrochloride (GuaHCl, G3272, Sigma-Aldrich) was used as an inhibitor of viral replication at 2mM.
DDD85646/IMP-366(2,6-dichloro-4-[2-(1-piperazinyl)-4-pyridinyl]-N-(1,3,5-trimethyl-1H-pyrazol-4-yl)-benzenesulfonamide) was purchased from Cayman Chemicals and used as an inhibitor of viral assembly at the final concentration of 5 µM. The cells were subsequently collected for Western blot, RNA isolations, plaque assays, or fixed with PFA (4%) for confocal imaging.
DDD85646/IMP-366(2,6-dichloro-4-[2-(1-piperazinyl)-4-pyridinyl]-N-(1,3,5-trimethyl-1H-pyrazol-4-yl)-benzenesulfonamide) was purchased from Cayman Chemicals and used as an inhibitor of viral assembly at the final concentration of 5 µM. The cells were subsequently collected for Western blot, RNA isolations, plaque assays, or fixed with PFA (4%) for confocal imaging.
10
Glial Co-Culture Model of Hyperammonemia
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The primary rat glial co-cultures of astrocytes containing 5–10% (M5, representing "physiological" conditions) or 30–40% (M30, representing "pathological" conditions) of microglia were incubated with 3 mM, 5 mM, 10 mM and 20 mM ammonium chloride (NH4Cl) (diluted in H2O) (Sigma-Aldrich, Germany) for 6 h and 24 h in 7% CO2 at 37 °C in order to mimic the conditions of HE. Because the survival of the glial cells after incubation with high concentration (20 mM) of NH4Cl can be affected, it was not used for experiments testing microglia morphology, Cx43 and AQP4 expression.
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