Eight to ten week old male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Animals were maintained in an environmentally controlled room with ad libitum access to both food and water. All procedures herein were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee before the onset of experimentation. Microcystin-LR (Cayman Chemical, Ann Arbor, MI) was diluted into PBS and administered at 120µg/kg i.p. to mice in the morning, whereupon mice were sacrificed after 45 minutes or 90 minutes by exsanguination and cervical dislocation. Alternately, some mice were given 700mg/kg galactosamine and 100µg/kg lipopolysaccharide i.p. as a separate model of established apoptosis (Jaeschke et al., 1998 (link)). Blood was stored on ice until centrifugation at 12,000 x g for 3 minutes to acquire plasma. Liver sections were snap frozen in liquid nitrogen for later usage. A portion of the liver was stored in buffered formalin for immunohistochemistry.
Microcystin lr
Manufactured by Cayman Chemical
Sourced in United States
Microcystin-LR is a laboratory standard used for analysis and research. It is a cyclic heptapeptide toxin produced by certain types of cyanobacteria. The primary function of Microcystin-LR is to serve as a reference material for the identification and quantification of this toxin in environmental and biological samples.
Automatically generated - may contain errors
Lab products found in correlation
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Accelerated solvent extractor, by Thermo Fisher Scientific (1 mentions)
Polysulfone psf, by Merck Group (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Sodium phosphate monobasic, by Merck Group (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Tris buffer, by Merck Group (1 mentions)
6 n hcl, by Merck Group (1 mentions)
Cacl2, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Fecl3, by Thermo Fisher Scientific (1 mentions)
5 protocols using microcystin lr
1
Induction of Apoptosis in Mice
2
Extraction and Analysis of Cyclopeptides from Mushroom Tissue
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For both the “Classic” and Accelerated Solvent Extractor (Thermo Fisher) protocol, the mushroom tissue was prepared in an identical fashion. Approximately 100 mg of dried mushroom tissue with the addition of 1 µg of microcystin‐LR (Internal standard; obtained from Cayman Chemical) was frozen with liquid nitrogen and subsequently macerated using a mortar and pestle. microcystin‐LR, a cyclic heptapeptide toxin produced by the blue‐green alga Microcystis aeruginosa (Eriksson et al., 1989 ), was added to all the extracted samples to confirm exhaustive extraction of cyclopeptides, which are chemically similar to this internal standard. The ground tissue was then prepared for extraction with either the accelerated solvent extraction (ASE) or “Classic” approach outlined below. For each analysis, three biological replicates of 100 mg of dried mushroom tissue were prepared independently and analyzed.
3
Polysulfone Membrane Fabrication Protocol
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Polysulfone (PSf) with a molecular weight of 35,000 g/moL, sodium phosphate monobasic, and sodium phosphate dibasic were obtained from Sigma Aldrich (St. Louis, MO, USA). ACS Grades N-Methyl-2-pyrrolidone (NMP), BDH1141-4LP, 95–98% concentrated sulfuric acid(H2SO4), BDH3070-25LPC, methanol, and acetic acid (glacial) were obtained from VWR BDH Chemicals (Solon, OH, USA). Polyether ether ketone (PEEK) pallets, 23969-50, were obtained from Polysciences Inc., (Warrington, PA, USA). Microcystin-LR was obtained from Cayman Chemical (Ann Arbor, MI, USA).
4
Biodegradation of Microcystin-LR Toxin
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Microcystin-LR was purchased from Cayman Chemicals, (Ann Arbor, Michigan, USA).
CaCl 2 and FeCl 3 was bought from Fisher Scientific, (Ontario, Canada). Millipore system (Milford, MA, USA) Milli-Q/Milli-RO was used to prepare mineral salt media (MSM) solutions spiked with MC-LR.
Sodium chloride (NaCl), peptone and yeast extract were purchased from Fisher Scientific (Ottawa, ON, Canada) and used to prepare Luria-Bertani medium for bacterial culture and inoculation of the isolated bacteria. For the toxicity assay: Tris-HCl buffer (pH 7.5) was prepared using Tris-buffer and 6N HCl (Merck, US) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT ) was used for measuring cell viability, bought from Sigma Aldrich, (Ontario, Canada). (Ishii et al., 2004) (link) and as a bioindicator for determining the toxicity of the biodegraded broth (Botsford et al., 1997) .
CaCl 2 and FeCl 3 was bought from Fisher Scientific, (Ontario, Canada). Millipore system (Milford, MA, USA) Milli-Q/Milli-RO was used to prepare mineral salt media (MSM) solutions spiked with MC-LR.
Sodium chloride (NaCl), peptone and yeast extract were purchased from Fisher Scientific (Ottawa, ON, Canada) and used to prepare Luria-Bertani medium for bacterial culture and inoculation of the isolated bacteria. For the toxicity assay: Tris-HCl buffer (pH 7.5) was prepared using Tris-buffer and 6N HCl (Merck, US) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT ) was used for measuring cell viability, bought from Sigma Aldrich, (Ontario, Canada). (Ishii et al., 2004) (link) and as a bioindicator for determining the toxicity of the biodegraded broth (Botsford et al., 1997) .
5
Microcystin-LR Cytotoxicity Assay
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Microcystin-LR was purchased from Cayman chemicals, (Ann Arbor, Michigan, USA). For measuring the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was brought from Sigma Aldrich, (Ontario, Canada). All the analytical reagents and chemicals used in preparing the culture media and other nutrient solutions were brought from Fisher Scientific, (Ontario, Canada).
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