Serum calcium was measured with the Calcium Colorimetric Assay kit (BioVision, Milpitas, CA, USA; K380-250). Serum phosphate was measured with the Phosphate Colorimetric Assay Kit (BioVision; K410-500). Brain/bone ACh and NE were measured by HPLC (Vanderbilt University Neurochemistry Core, Nashville, TN, USA) as described in.(41 (link),42 (link))
Phosphate colorimetric assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Phosphate Colorimetric Assay Kit is a laboratory tool used to quantify phosphate (PO4) levels in various samples. The kit utilizes a colorimetric reaction to detect and measure phosphate concentration. The assay provides a simple, accurate, and fast method for determining phosphate levels in biological, environmental, and other relevant samples.
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Lab products found in correlation
Calcium colorimetric assay kit, by Abcam (4 mentions)
Ratlaps eia kit, by Immunodiagnostic Systems (2 mentions)
Synergy ht spectrophotometer, by Agilent Technologies (2 mentions)
Alp colorimetric assay kit, by Abcam (1 mentions)
Ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Infinite m1000 pro microplate reader, by Tecan (1 mentions)
Triglyceride quantification kit, by Cell Biolabs (1 mentions)
Bilirubin assay kit, by Cell Biolabs (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Alkaline phosphatase activity fluorometric assay kit, by Abcam (1 mentions)
Mouse osteocalcin eia kit, by Biomedical Technologies (1 mentions)
Ctx 2, by Immunodiagnostic Systems (1 mentions)
Ctx 1, by Immunodiagnostic Systems (1 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
2414 refractive index detector, by Waters Corporation (1 mentions)
Hi 2210 ph meter, by Hanna Instruments (1 mentions)
7700 series icp ms, by Agilent Technologies (1 mentions)
Calcium liquicolor test kit, by EKF Diagnostics (1 mentions)
Prism v5, by GraphPad (1 mentions)
Sphingosine 1 phosphate (s1p), by Merck Group (1 mentions)
28 protocols using phosphate colorimetric assay kit
1
Serum Calcium, Phosphate, and Neurotransmitter Analysis
2
Enzymatic Activities of Recombinant Parasites
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ATP and ADP hydrolysis activities of live parasites, or intact CHO-S cells (expressing recombinant SmATPDase1), or mock transfected cells, were determined as described above with slight modifications. Briefly, live parasites, or CHO-S cells, were first washed 3 times in isotonic wash solution (20 mM HEPES buffer, pH 7.4 containing 0.13 M NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM Glucose). Next, a specific number of parasites or cells were resuspended in 100 µl isotonic wash solution. Reactions were started by the addition of a 100 µl of the same buffer containing ATP or ADP to produce a final concentration of 2 mM. Released inorganic phosphates were measured using the Phosphate Colorimetric Assay Kit (BioVision) according to the manufacturer’s instructions.
3
Quantifying Cell Cytotoxicity and Biomarkers
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The levels of lactate dehydrogenase (LDH) in supernatants were measured by using the LDH Cytotoxicity Assay Kit according to manufacturer instructions (Thermo Fisher Scientific, Waltham, MA, USA). The assay relies on conversion of lactate to pyruvate via NAD+ reduction to NADH by LDH. Diaphorase then uses NADH to reduce a tetrazolium salt (INT) to a red formazan product that can be measured at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the medium, which is indicative of cytotoxicity. Absorbance at 490 and 680 nm was measured using a Tecan infinite M1000 pro microplate Reader (Tecan, Männerdorf, Switzerland). Alkaline phosphatase (ALP) content was measured using the ALP colorimetric assay kit, according to manufacturer’s instructions (BioVision, Milpitas, CA, USA). The kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate, which turns yellow (405 nm) when dephosphorylated by ALP. Phosphate content was measured using the phosphate colorimetric assay kit, according to manufacturer’s instructions (BioVision, CA, USA). The assay utilizes a formulation of malachite green and ammonium molybdate, which forms a chromogenic complex with phosphate ions. The absorption of the latter complex was measured at 650 nm.
4
Gallstone Biochemical Composition Analysis
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We used the following assay kits to determine the components of gallstones; Cholesterol Enzymatic Assay Kit (Xpressbio, Fredrick, MD) for cholesterol, Triglyceride Quantification Kit (Cell Biolabs, San Diego, CA) for triglycerides, Bilirubin Assay Kit (Cell Biolabs) for bilirubin, and Phosphate Colorimetric Assay Kit (Biovision, Mountain View, CA) for bilirubin. The gallstones were homogenized with the substances described in each assay kit and the supernatant was collected for assay. After incubation for a determined period at 37 °C in the assay buffer, the absorbance was measured using a microplate reader (model 680; Bio-Rad, Hercules, CA).
5
Quantifying Intracellular and Extracellular Phosphate
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Extra- and intracellular phosphate concentration was determined by Phosphate Colorimetric Assay Kit (BioVision). Extracellular phosphate was measured from the culture medium. Cells were treated with 0.1 M NaOH and 0.1% SDS to measure intracellular phosphate. Quantification of phosphate was measured with an OD at 650 nm by spectrophotometry. The results were normalized to the total protein concentration, which was measured in the cultures using a protein assay reagent (Bio-Rad), based on the Bradford dye binding procedure, and albumin was used as a standard. Each condition was performed in triplicate and expressed as nmol phosphate/µg protein.
6
Serum Markers of Bone Turnover
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We determined the activity of osteoblast and osteoclast cells using serum indicators rather than histomorphometry. Serum concentrations of alkaline phosphatase (ALP), osteocalcin, procollagen I C-terminal propeptide (PICP), pyridinoline (PYD), cross-linked N-telopeptide of type I collagen (NTXI) were all measured using commercial kits according to manufacturer's instructions. (ALP: Alkaline phosphatase activity fluorometric assay kit (Biovision, Mountain View, CA, USA); osteocalcin: Mouse osteocalcin EIA kit (Biomedical Technologies, Stoughton, MA, USA); PICP: ELISA kit for mouse PICP (USCN Life Science, Wuhan, Hubei, China); PYD: Mouse pyridinoline ELISA kit (CUSABIO, Wuhan, Hubei, China); NTXI: ELISA kit for mouse NTXI (USCN Life Science, Wuhan, Hubei, China)). Calcium was determined with a calcium colorimetric assay kit (Biovision, Mountain View, CA, USA), and phosphate was measured with a phosphate colorimetric assay kit (Biovision, Mountain View, CA, USA).
7
Serum Biomarkers of Bone Metabolism in Mice
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Blood was collected from mice by cardiac puncture for analysis of several biochemical parameters. Serum total calcium was determined using Stanbio Laboratory Calcium Liquidcolor (CPC; Boerne, TX, USA; Catalog No. 0150‐250). Serum inorganic phosphate was determined using a Phosphate Colorimetric Assay Kit (BioVision, Inc., Milpitas, CA, USA; Catalog No. K410‐500). Serum C‐terminal telopeptides of types I and II collagen (CTX‐1 and CTX‐2; Immunodiagnostic Systems Inc., Fountain Hills, AZ, USA; Catalog No. AC‐06F1 and AC‐08F1, respectively), as well as N‐terminal propeptide of type I procollagen (P1NP) were determined using the RatLaps EIA kit (Immunodiagnostic Systems Inc., Fountain Hills, AZ, USA; Catalog No. AC‐33F1). Serum intact mouse parathyroid hormone (PTH(1‐84)) was measured using the mouse PTH(1‐84) EIA (Quidel Corp, San Diego, CA, USA; Catalog No. 60‐2305).
8
NAD Cleavage Assay for rSmNPP5
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Recombinant SmNPP5, expressed in suspension adapted FreeStyle Chinese Hamster Ovary Cells (CHO-S), was purified from culture medium by standard immobilized metal affinity chromatography (IMAC) using HisTrap Excel columns, as described previously [12 ]. The ability of rSmNPP5 to cleave NAD was measured by two methods. In the first method, recombinant enzyme (12.5–50ng) was incubated with ε-NAD essentially as described above but at room temperature (RT). The second method employed β-NAD (Roche, 10127965001); after incubating 1 μg SmNPP5 with β-NAD (2 mM) in 500 μl enzyme assay buffer for either 1- or 5-hours at 37°C, calf intestinal alkaline phosphatase (CIP, at 70 u/mL, Promega, M182A) was subsequently added to samples of each reaction mixture for 15 min at RT. Any inorganic phosphate (Pi) released was then measured using a Phosphate Colorimetric Assay Kit (BioVision, K410), following the manufacturer’s instructions. Absorbance (650 nm) was measured using a Synergy HT microplate reader (Biotek).
9
Measurement of Intracellular Phosphate in AEC2s
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Mice were sacrificed by injection with an overdose of Avertin and lavaged with 1 mL of sterilized saline through a 20-gauge angiocatheter inserted into the trachea. BALF from patients with PF or from patients without chronic inflammatory lung diseases were obtained from Beijing Friendship Hospital (Supporting Information Table S1 ). All the subjects gave appropriate informed consent to participate in this study. The study design was approved by the institutional ethical committee. Briefly, 50 mL of sterilized saline at body temperature was instilled through the bronchoscope. Mucus was removed from the fluid by filtration through two sheets of gauze. The BALF was centrifuged for 10 min at 400 × g at 4 °C. Phosphate concentration was measured with a Phosphate Colorimetric Assay Kit (BioVision) following the manufacturer's instructions. For intracellular phosphate determination, 1 × 106 AEC2s were washed with cold saline twice and suspended in 1 mL of double-distilled water. The suspension was sonicated for 30 s six times at 10 s intervals. The samples were centrifuged for 15 min at 4 °C at 12,000 × g, and intracellular phosphate in the supernatant was measured.
10
Quantifying REE, Th, and Biomass in Bioreactors
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Rare earth element, Th, phosphate, glucose, pH, and biomass were quantified by previously described analytical methods (Brisson et al., 2016 (link)). Briefly, REE and Th concentrations were measured using an Agilent Technologies 7700 series ICP-MS. Phosphate concentration was measured using the BioVision Phosphate Colorimetric Assay Kit. Glucose was measured by HPLC on a Waters 2695 HPLC system with a BioRad Aminex HPX-87H carbohydrate/organic acids analysis column and a Waters 2414 refractive index detector. pH was measured using a Hanna Instruments HI 2210 pH meter. Biomass was measured as total volatile solids of filter-collected samples by drying at 105°C and subsequent ashing at 550°C as described previously (Brisson et al., 2016 (link)) based on United States Environmental Protection Agency Method 1684 (US EPA, 2001 ). REE, Th, phosphate, glucose, and pH measurements were taken for six biological replicates for each time point (0, 2, 4, and 6 days after inoculation), while biomass measurements were taken for three biological replicates at time points 2, 4, and 6 days.
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