MPA (J&K China Chemical Ltd.); Mn(Ac)2·4H2O, Zn(Ac)2·2H2O, Na2S·9H2O (Tianjin Kemiou Chemical Co., China); EDC, NHS, MC-LR (Sigma, US) were used in the experiments. Malachite green (MG), microcystin-RR (MC-RR), and paraquat (PQ) were bought from Shanghai Sangon Biotechnology Co. Ltd. (China). All other reagents were of analytical purity.
Mc lr
Manufactured by Merck Group
Sourced in United States, France
MC-LR is a laboratory instrument manufactured by Merck Group. It is designed for the detection and quantification of the toxin microcystin-LR in various water samples. The core function of MC-LR is to provide accurate and reliable analytical data to support environmental monitoring and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mc rr, by Merck Group (5 mentions)
Mc yr, by Merck Group (5 mentions)
Mc lw, by Merck Group (3 mentions)
Mncl2, by Sinopharm (2 mentions)
Bovine serum albumin (bsa), by Sinopharm (2 mentions)
Costar 9018, by Corning (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sep pak c18 cartridge, by Waters Corporation (2 mentions)
Formic acid, by Merck Group (1 mentions)
Ammonium hydroxide solution nh4oh, by Merck Group (1 mentions)
Strata x cartridge, by Phenomenex (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Gold 3 chloride hydrate, by Merck Group (1 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride edc, by Merck Group (1 mentions)
15 protocols using mc lr
1
Sensitive Analyte Detection Protocol
2
Microcystin Quantification in Water
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All chemicals were reagent grade or higher in quality. Formic acid (FA) and ammonium hydroxide solution (NH4OH) were purchased from Sigma-Aldrich (Milan, Italy). HPLC gradient grade methanol (MeOH), acetonitrile (ACN) and ultrapure water (H2O) were supplied by Carlo Erba (Cornaredo, Italy). Strata™-X cartridges (200 mg, 3 mL) were purchased from Phenomenex (Castel Maggiore, Italy). DA (≥90%), OA sodium salt (MQ 100) and analytical grade standard MC-RR, MC-LR, MC-YR and MC-LW solutions were supplied by Sigma-Aldrich (Milan, Italy). Individual stock standard solutions of all analytes (1 μg mL−1) were prepared in MeOH, stored in the dark (−20 °C) and renewed weekly.
3
Cyanotoxin and Nutrient Analysis Protocol
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Three 100 ml aliquots from each dialysis bag were immediately placed on ice and returned to the lab for filtration on a 0.7 μm Whatman GF/F glass microfiber filter (Fisher Scientific cat # 09-874-64) and stored at −20 °C for cyanotoxin analysis. According to Fastner et al.63 and Dyble et al.64 (link), toxin samples were lyophilized first and then sonicated in 75% aqueous methanol. MC analogues (MC-LR, MC-RR, MC-YR, MC-LA; Sigma-Aldrich) and cylindrospermopsin (CYN) (Sigma-Aldrich) analysis was performed by High-Performance Liquid Chromatography coupled Mass Spectrometry (HPLC/MS) using a Thermo Surveyor MSQ Single Quadrupole Mass Selective Detector and Thermo Spectrasystem gradient chromatographic system according to a method described by Barco et al.65 (link). Total MC concentrations were reported as the sum of all congeners (HPLC/MS-Total).
Total Kjeldahl nitrogen (TKN-N) and ammonia (NH3-N) were analyzed on a BRAN+LUEBBE Autoanalyzer66 . Nitrate (NO3-N), total phosphorus (TP-P), and soluble reactive phosphorus (SRP-P) were analyzed on an ion chromatograph (detections limit: 0.005 mg/L, Standard Methods 4100 C)67 .
Total Kjeldahl nitrogen (TKN-N) and ammonia (NH3-N) were analyzed on a BRAN+LUEBBE Autoanalyzer66 . Nitrate (NO3-N), total phosphorus (TP-P), and soluble reactive phosphorus (SRP-P) were analyzed on an ion chromatograph (detections limit: 0.005 mg/L, Standard Methods 4100 C)67 .
4
Biodegradation of Microcystin-LR by Brevibacillus
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Acetonitrile, MCLR (98.5%), and trifluoroacetic acid were obtained from Sigma (Saint-Quentin Fallavier, France). PP1 (1200 U/mL) was purchased from EMD Millipore (Darmstadt, Germany). Bovine serum albumin, beef extract peptone medium, MnCl2, p-nitrophenyl disodium orthophorphate, tris(hydroxymethyl)aminomethane, and other reagents were purchased from Sinopharm (Shanghai, China).
Toxic M. aeruginosa FACHB-905 (producing MCLR) was grown in BG11 medium at 25 °C with a light/dark cycle (12/12). The cultures were harvested at the late exponential phase of growth and had a final cell yield up to about 107 cells/mL [17 (link)]. The biodegradation bacterium Brevibacillus sp. D1 (GenBank code EU593881), which could effectively remove algae and MCLR, was kindly supplied by Professor Ruimin Mu at Shandong Jianzhu University. Brevibacillus sp. D1 was grown in beef extract peptone medium at 35 °C and harvested at OD420 nM ≈ 1.0 (109 cells/mL).
Toxic M. aeruginosa FACHB-905 (producing MCLR) was grown in BG11 medium at 25 °C with a light/dark cycle (12/12). The cultures were harvested at the late exponential phase of growth and had a final cell yield up to about 107 cells/mL [17 (link)]. The biodegradation bacterium Brevibacillus sp. D1 (GenBank code EU593881), which could effectively remove algae and MCLR, was kindly supplied by Professor Ruimin Mu at Shandong Jianzhu University. Brevibacillus sp. D1 was grown in beef extract peptone medium at 35 °C and harvested at OD420 nM ≈ 1.0 (109 cells/mL).
5
Immunoassay for Detecting Okadaic Acid
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Gold(III) chloride hydrate, DA, OA, MC-LR, soybean trypsin inhibitor (STI), bovine serum albumin (BSA), methanol, sucrose, sodium azide, Triton X-100, dimethyl sulfoxide (DMSO), 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB), N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride (EDC) were from Sigma-Aldrich (Saint Louis, MO, USA). Goat anti-mouse and donkey anti-goat immunoglobulins (GAMI and DAGI) were from Arista Biologicals (Allentown, PA, USA). Peroxidase-labeled GAMI (GAMI–HRP) were from Jackson Immuno Research Labs (West Grove, PA, USA). MAbs against OA (clone 7E1) were from Santa Cruz Biotechnology (Dallas, TX, USA). According to the manufacturer, the MAbs have strict specificity to OA and do not interact with its structural analogs, such as dinophysistoxin-1, dinophysistoxin-2, dinophysistoxin-3, and pectenotoxin-2. All other compounds were analytically pure. Polystyrene 96-well transparent microplates Costar 9018 from Corning Costar (Tewksbury, MA, USA) were used for the ELISA.
6
Enzymatic Phosphorylation Assay
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MCLR, MCLF, MCLA, MCLY, and MCLW were purchased from Sigma (Saint-Quentin Fallavier, France). PP2A was purchased from New England Biolabs Inc (Beverly, MA, USA). Bovine serum albumin, dithiothreitol, MnCl2, P-nitrobenzene disodium orthophosphate, sodium thiosulfate, tris(hydroxymethyl) aminomethane, and other reagents were purchased from Sinopharm (Shanghai, China).
7
Indirect ELISA for Okadaic Acid Detection
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OA, domoic acid (DA), MC-LR, brevetoxin (BTX), soybean trypsin inhibitor (STI), bovine serum albumin (BSA), N,N′-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC), fluorescein 5(6)-isothiocyanate (FITC), methanol, chloroform, dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA), Triton X-100, and trimethylamine were acquired from Sigma-Aldrich (Saint Louis, MO, USA). Ethylenediamine dihydrochloride and 5-(aminomethyl) fluorescein hydrochloride (AMF) were acquired from ThermoFisher Scientific (Waltham, MA USA). A 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB) substrate solution was purchased from Immunotekh (Moscow, Russia). Goat anti-mouse immunoglobulins labeled with horseradish peroxidase (GAMI–HRP) were obtained from Jackson Immuno Research Labs (West Grove, PA, USA). Monoclonal antibodies (MAbs) against OA (clone 7E1) were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). All other compounds were analytically pure. For the ELISA test, polystyrene 96-well transparent microplates Costar 9018 from Corning Costar (Tewksbury, MA, USA) were used.
8
Cyanotoxin Mixture Cytotoxicity Assay
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Cyanotoxins used in this work were used alone and in two mixtures (group A and group B). See Table 1 for chemical formulas and for the brief description of each toxin. CYN, MC-LR, and ATX-a were purchased from Enzo Life Sciences, Inc. (NY, USA). MC-LR and CYN were dissolved in sterile water to 1 mM. ATX-a was dissolved in sterile water to 2.2 mM. BMAA and DAB were obtained from Sigma–Aldrich Canada Co. (ON, Canada) and dissolved in sterile water to 65 mM. All solutions were stored at −80 °C until use. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma–Aldrich Canada Cie. (ON, Canada). MTT was prepared as a 5 mg/ml stock solution in phosphate-buffered saline (PBS 1X), filtered through a 0.22 μm filter before use, and diluted to 1 mg/ml in a serum-containing medium. Mouse TNF-α ELISA assay kit was purchased from Thermo Fisher Scientific Company (ON, Canada). Caspase-Glo 3/7Aassay kit was purchased from Promega Corporation (WI, USA). All other compounds and reagents, were obtained from Wisent, Inc. (QC, Canada).
9
Methylation of Microcystin Variants
10
Microcystin-LR Treatment of C. reinhardtii
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The C. reinhardtii wild-type strain was purchased from the Freshwater Algae Culture Collection Center at the Institute of Hydrobiology (Chinese Academy of Sciences (CAS), Wuhan, China). Cells were cultured under continuous light at 23°C in Tris-acetate-phosphate (TAP) medium. MC-LR was purchased from the Sigma Chemical Company (St. Louis, MO, USA). For MC-LR treatment, C. reinhardtii cells in the stationary phase (106 cells/ml) were treated with different concentrations of MC-LR. At different time points, cells were removed for further analysis.
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