Primary feline monocytes (2 × 105 cells) were cultured in medium containing chloroquine (Wako Pure Chemical Industries, Japan) or ammonium chloride (Wako Pure Chemical Industries, Japan) in 24-well multi-plates at 37 °C with 5% CO2 for 1 h. After washing with PBS, FIPV 79-1146 or FECV 79-1683 (1 × 104 TCID50 for both) with or without pretreatment with mAb 6-4-2 was added to the culture and allowed to adsorb to the cells at 37 °C with 5% CO2 for 1 h in the presence of chloroquine or ammonium chloride. After washing with PBS, the cells were cultured in the medium containing chloroquine or ammonium chloride, and the culture supernatants were collected after 48 h. The virus titer in the culture supernatant (TCID50) was determined by the method of Reed and Muench [22 ] with fcwf-4 cells. To evaluate the cytotoxic effects of chloroquine and ammonium chloride in primary feline monocytes, cell viability was measured by the WST-8 assay as described before [29 (link)].
Ammonium chloride
Manufactured by Fujifilm
Sourced in Japan
Ammonium chloride is a chemical compound that is commonly used in various laboratory applications. It is a white, crystalline solid with a chemical formula of NH4Cl. Ammonium chloride serves as a key ingredient in various laboratory reagents and buffers, where its primary function is to provide an ammonium ion source and adjust the pH of solutions.
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Lab products found in correlation
Chloroquine, by Fujifilm (1 mentions)
Isoflurane, by Abbott (1 mentions)
Male balb c mice, by Charles River Laboratories (1 mentions)
Recombinant mouse il 15, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Fujifilm (1 mentions)
Potassium chloride (kcl), by Fujifilm (1 mentions)
Pi for flow cytometry, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Sodium chloride (nacl), by Fujifilm (1 mentions)
Opti modified eagle s medium opti mem, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Fujifilm (1 mentions)
Monothioglycerol, by Nacalai Tesque (1 mentions)
Mem non essential amino acids, by Nacalai Tesque (1 mentions)
Penicillin streptomycin glutamine mixed solution, by Nacalai Tesque (1 mentions)
Rpmi 1640 medium, by Nissui Pharmaceutical (1 mentions)
Hydrogen peroxide, by Fujifilm (1 mentions)
Sodium hydroxide (naoh), by Fujifilm (1 mentions)
5 protocols using ammonium chloride
1
Chloroquine and Ammonium Chloride Effects on Feline Monocytes Infected with FIPV and FECV
2
Ammonium Chloride-Induced Energy Metabolism in Mice
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Male BALB/c mice (30-weeks old; Charles River Laboratories) were maintained as described in Experiment 1. Mice were intraperitoneally administered ammonium chloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan) solution (100 mg/kg-BW) 3 times a week for 4 weeks23 (link). The same amount of saline was administered to negative control animals. During the experimental period, mice were fed CE-2 (CLEA Japan) and water was provided ad libitum. After 4 weeks, energy metabolism was analysed by indirect calorimetry (see Indirect calorimetry analysis); then the mice were anesthetised with isoflurane (Abbott Japan, Tokyo, Japan) and the gastrocnemius muscle was resected and stored at −80 °C.
3
Biochemical Reagent Preparation Protocol
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Ammonium chloride (NH 4 Cl), Tris-hydroxymethyl aminomethane (Tris-HCl) and glycerol were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Bovine serum albumin (BSA) was purchased from Nacali Tesque (Kyoto, Japan). Recombinant mouse IL-15 was purchased from PeproTech (Rocky Hill, NJ).
4
Cell Apoptosis Evaluation Protocol
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RPMI 1640 medium was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Opti-modified Eagle’s medium (Opti-MEM) and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Sodium chloride, sodium bicarbonate, potassium chloride, Tris-HCl, ethylenediaminetetraacetic acid (EDTA), ammonium chloride, and glucose were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Monothioglycerol, MEM non-essential amino acids, and penicillin-streptomycin-glutamine mixed solution were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The 20 bp ladder was purchased from Takara Bio (Otsu, Japan). Propidium iodide, R848, and ovalbumin were obtained from Merck KGaA (Darmstadt, Germany). Alexa Fluor 488 annexin V/ Dead Cell Apoptosis kit with Alexa Fluor 488 annexin V and PI for Flow Cytometry was purchased from Invitrogen (San Diego, CA, USA). All other chemicals were of the highest grade available and were used without further purification.
5
HPLC-PR-CL Method for NDMA Quantification
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Analytical-grade NDMA solution with concentration of 100 mg/L was purchased from Ultra Scientific (Kingstown, RI, USA) and used as the standard for the HPLC-PR-CL method. An NDMA stock solution was prepared at 1 mg/L in pure methanol. Luminol (5-amino-2,3dihydro-1,4-phthalazinedione) from Wako Pure Chemical Industries (Tokyo, Japan) was used for HPLC-PR-CL. A Luminol stock solution was prepared at 20 mM in a 0.5 M carbonate buffer. Hydrogen peroxide, sodium hypochlorite (to represent hypochlorite), ammonium chloride and sodium hydroxide were of analytical grade (Wako Pure Chemical Industries, Tokyo, Japan). These four chemicals were used to evaluate their influence on HPLC-PR-CL.
Ascorbic acid and sodium thiosulfate (Wako Pure Chemical Industries, Tokyo, Japan) were used to quench chloramine in treated wastewater. Treated wastewater was collected from the permeate stream of a pilot-scale UF system housed at a municipal wastewater treatment plant (WWTP) in Japan, where the secondary effluent is fed to the UF system. The wastewater treatment consisted of screen, sedimentation and bioreactor processes. The pilot-scale UF system was equipped with one HFU-2020 membrane module (Toray Industries, Inc., Tokyo, Japan) with a nominal pore size of 0.01 µm.
Ascorbic acid and sodium thiosulfate (Wako Pure Chemical Industries, Tokyo, Japan) were used to quench chloramine in treated wastewater. Treated wastewater was collected from the permeate stream of a pilot-scale UF system housed at a municipal wastewater treatment plant (WWTP) in Japan, where the secondary effluent is fed to the UF system. The wastewater treatment consisted of screen, sedimentation and bioreactor processes. The pilot-scale UF system was equipped with one HFU-2020 membrane module (Toray Industries, Inc., Tokyo, Japan) with a nominal pore size of 0.01 µm.
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