Urinary and plasma levels of creatinine, urinary γ-glutamyl transferase (γ-GT) activity and proteinuria were determined by spectrophotometry (Turner SP-830 plus Barnstead, Dubuque, Iowa, USA) using commercially available kits (BioClin/Quibasa, Belo Horizonte, Brazil). Plasma and urinary concentrations of Na+ and K+ were measured by flame photometry (CELM 180; Belo Horizonte, Minas Gerais, Brazil). Osmolality was determined in plasma and urine samples by cryoscopic osmometery using a Micro-Osmometer 3320 (Advanced Instruments, Norwood, Massachusetts, USA). Urine proteins were also analyzed by gel electrophoresis which was performed according to Laemmli (1970) (link). Urine samples from animals of different times post-venom injection were diluted (10 ×) and aliquots of 10 μL were submitted to SDS-PAGE on 8-20 % gradient gels under reducing conditions. Toxins excreted in urine were detected by Western-blot as previously described (Pinto et al., 2006 (link)). Aliquots containing 50 μg of protein were separated by SDS-PAGE and transferred to PVDF membranes. Toxins were recognized using as a primary antibody an equine anti-LOBE IgG (ALS) diluted 1:100 and as a secondary antibody a peroxidase-labeled anti-horse IgG diluted 1:1000.
3320 micro osmometer
Manufactured by Advanced Instruments
Sourced in United States
The 3320 Micro Osmometer is a laboratory instrument designed to measure the osmolality of small sample volumes. It utilizes the freezing point depression method to determine the osmotic concentration of a solution.
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5 protocols using 3320 micro osmometer
1
Toxin Excretion Monitoring in Envenomation
2
Urine and Plasma Biochemistry Analysis
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Collected urine was centrifuged at 1000 g for 1 min to clear sediments. Osmolality was measured on a Micro Osmometer 3320 (Advanced Instruments Inc). Blood plasma was prepared by collecting blood from the retro-orbital plexus in Li-heparin tubes, inverting 8–10 times, followed by centrifugation. Urine and plasma sodium, chloride, potassium, urea, and creatinine concentrations were analyzed by the Clinical Pathology Laboratory at the Medical Research Council (Harwell). Plasma aldosterone concentrations were determined using an enzyme immunoassay kit (EIA-5298; range: 20–1000 pg/ml; QC: standards, DRG International).
3
Comprehensive Biological Measurements Protocol
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Urine osmolality was measured using a freezing point depression osmometer (3320 Micro Osmometer; Advanced Instruments). Serum samples were analyzed at Aarhus University Hospital, Skejby for cholesterol measurement. Total cholesterol, high‐density lipoprotein (HDL), and triglycerides were measured directly and low‐density lipoprotein (LDL) were calculated using the Friedewald formula. Sodium and potassium concentrations were measured by flame photometry (420 Flame Photometer; Sherwood Scientific Ltd).
4
Preparation and Characterization of Mannose 6-Phosphate Solutions
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Mannose 6-Phosphate (M6P; Sigma-Aldrich, UK) was originally prepared for study using 14 mg/ml, 56 mg/ml and 169 mg/ml to produce 50 mM, 200 mM, and 600 mM solutions respectively.
Mannose 6-Phosphate (M6P; Sigma-Aldrich, UK), or Glucose 6-Phosphate (G6P; Sigma-Aldrich, UK) was weighed to make up a 600 mM (169.26 mg/mL) solution, which was then placed into a volumetric flask and Phosphate buffered saline (PBS, Life Technologies, USA.) added (approximately 2 mL less than the total desired volume was prepared to allow for neutralisation). The solution was inverted several times to aid dissolution. A 100 µL pipette (Gilson, UK) was used to slowly add 10M Sodium Hydroxide (NaOH; Sigma-Aldrich, UK) drop wise to the solution, swirling after each addition, until the solution was neutralised. The solution was allowed to stand at room temperature for 30 min to allow any remaining M6P or G6P to dissolve. After 30 minutes, the pH of the solution was determined and adjusted to pH 7.0 (±0.5) using 10M NaOH. From this stock solution dilutions were made to prepare 50 mM, 200 mM and 600 mM solutions using PBS.
In subsequent studies osmolality was checked at 150 mM, 300 mM and 600 mM using a 3320 Micro-osmometer (Advanced Instruments Inc, USA.) and preparations specifically of 50 mM, 200 mM and 600 mM (Adaprev) were used for study.
Mannose 6-Phosphate (M6P; Sigma-Aldrich, UK), or Glucose 6-Phosphate (G6P; Sigma-Aldrich, UK) was weighed to make up a 600 mM (169.26 mg/mL) solution, which was then placed into a volumetric flask and Phosphate buffered saline (PBS, Life Technologies, USA.) added (approximately 2 mL less than the total desired volume was prepared to allow for neutralisation). The solution was inverted several times to aid dissolution. A 100 µL pipette (Gilson, UK) was used to slowly add 10M Sodium Hydroxide (NaOH; Sigma-Aldrich, UK) drop wise to the solution, swirling after each addition, until the solution was neutralised. The solution was allowed to stand at room temperature for 30 min to allow any remaining M6P or G6P to dissolve. After 30 minutes, the pH of the solution was determined and adjusted to pH 7.0 (±0.5) using 10M NaOH. From this stock solution dilutions were made to prepare 50 mM, 200 mM and 600 mM solutions using PBS.
In subsequent studies osmolality was checked at 150 mM, 300 mM and 600 mM using a 3320 Micro-osmometer (Advanced Instruments Inc, USA.) and preparations specifically of 50 mM, 200 mM and 600 mM (Adaprev) were used for study.
5
Measuring Polymer Solution Osmolality
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Polymer solutions were prepared as described by dissolution into DMEMþ10% FBS. The osmolality of the solutions was measured using a 3320 MicroOsmometer (Advanced Instruments, Inc. Norwood, MA), which measures the freezing point to determine solute concentration.
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