Urinary and plasma K+ concentrations were measured using Jenway PFP7 Flame photometer (Bibby Scientific). Plasma was separated by centrifugation at 1300 g in Vacutainer Plus SST plastic tubes with clot activator and gel for plasma separation (BD; Cat. # 367988). Urinary pH was measured using MI‐410 pH microelectrode (Microelectrodes Inc.). Urinary creatinine concentration was assessed with QuantiChrom Creatinine Assay Kit (BioAssay Systems; Cat. # DICT‐500) utilizing improved Jaffe method (Mamenko et al., 2012 (link)). Aldosterone was measured using an enzymatic immunoassay kit (Cayman Chemical; Cat. # 501090) in accordance with the vendor's protocol. Kidney injury marker 1 (KIM1) was measured using a commercially available kit (R&D System; Cat. # RKM100).
Jenway pfp7 flame photometer
Manufactured by Cole-Parmer
Sourced in United Kingdom
The Jenway PFP7 Flame Photometer is a laboratory instrument used for the determination of alkali and alkaline earth metal ions, such as sodium, potassium, calcium, and lithium, in aqueous solutions. The device operates by measuring the intensity of the characteristic emission spectra produced when the sample is atomized in a flame.
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Lab products found in correlation
Quantichrom creatinine assay kit, by BioAssay Systems (2 mentions)
Triton x 100, by Merck Group (2 mentions)
No 1 filter paper, by Cytiva (2 mentions)
Enzymatic immunoassay kit, by Cayman Chemical (1 mentions)
Microhematocrit capillary tube, by Thermo Fisher Scientific (1 mentions)
Quantichrom creatinine assay kit dict 500, by BioAssay Systems (1 mentions)
Model 3320, by Advanced Instruments (1 mentions)
Lactate dehydrogenase activity assay kit, by Cambridge Bioscience (1 mentions)
9 protocols using jenway pfp7 flame photometer
1
Urinary and Plasma Electrolyte Analysis
2
Assessing Sodium Balance Regulation
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Mice were acclimated for 3 days in metabolic cages (Techniplast, 3600M021) with free access to water and regular rodent chow (0.32% Na+; Envigo,TD.7012). Following acclimation, 24-hour urine samples were collected and assessed. As necessary, mice were fed with a Na+-deficient diet (< 0.01% Na+; Envigo, TD.90228) for at least 2 days or longer (up to 7 days) as described in the experimental protocols. Urinary creatinine concentration was assessed with QuantiChrom Creatinine Assay Kit (BioAssay Systems, DICT-500) utilizing improved Jaffe method (52 (link)). Aldosterone was measured using an enzymatic immunoassay kit (Cayman Chemical, 501090) in accordance with the vendor’s protocol. Urinary and plasma Na+ and K+ concentrations were measured using Jenway PFP7 Flame photometer (Bibby Scientific).
ENaC-induced natriuresis and anti-kaliuresis were assessed as the difference in respective urinary Na+ and K+ levels in sodium restricted Epac WT, Epac1–/–, Epac2–/–, and Epac1&2–/– mice for 6 hours before and following i.p. injection of amiloride (2 mg/kgBW). For i.p. injections of Epac1&2 inhibitor in mice, ESI-09 was freshly prepared in sterile 10% ethanol/Tween 80 and 90% PBS in concentration of 10 mg/mL and the injection volume was proportional to the animal weight.
ENaC-induced natriuresis and anti-kaliuresis were assessed as the difference in respective urinary Na+ and K+ levels in sodium restricted Epac WT, Epac1–/–, Epac2–/–, and Epac1&2–/– mice for 6 hours before and following i.p. injection of amiloride (2 mg/kgBW). For i.p. injections of Epac1&2 inhibitor in mice, ESI-09 was freshly prepared in sterile 10% ethanol/Tween 80 and 90% PBS in concentration of 10 mg/mL and the injection volume was proportional to the animal weight.
3
Measurement of Urinary Parameters in Water-Deprived Mice
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Mice were acclimated for few days in metabolic cages (3600M021; Techniplast, West Chester, PA, USA). Following acclimation and baseline measurements of urinary parameters, mice were water deprived for 24 hours, as we did previously [25 (link)]. Tails were clipped with sterilized scissors to collect 50 μl blood into heparinized micro-hematocrit capillary tubes (Fischer, Cat. # 22-362-566) before and after water deprivation. Urine and plasma osmolarity was measured in duplicates using freezing point depression osmometer Model 3320 (Advanced Instruments, Norwood, MA, USA). Urinary creatinine concentration was assessed with QuantiChrom Creatinine Assay Kit (DICT-500; BioAssay Systems, Hayward, CA, USA) utilizing the improved Jaffe method, as we did before [30 (link)]. Urinary AVP levels were assayed with Vasopressin Arg8-Vasopressin ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) following the manufacturer’s protocols. Urinary Ca2+ concentration was measured using a Jenway PFP7 Flame photometer (Bibby Scientific, Burlington, NJ, USA).
4
Potassium Leakage Assay for Pore-Forming Toxins
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The leakage of potassium was measured as described previously (17 (link), 29 (link)). Briefly, 1.5 × 105 cells/well were cultured for 24 h in complete medium using 6-well culture plates, and then treated with 27-hydroxycholesterol or 25-hydroxycholesterol for 24 h using the concentrations specified in Results. The cells were washed and challenged in potassium-free buffer for 5 min with the amounts of pyolysin or streptolysin O indicated in Results, or for 15 min with 8 µg α-hemolysin (no potassium leakage was detected after 5 min of α-hemolysin challenge). Extracellular potassium was measured in cell supernatants using a Jenway PFP7 flame photometer (Cole-Parmer, Stone, Staffordshire, UK). The inter- and intra-assay coefficients of variation were < 4%.
5
Measuring Cellular Potassium Leakage
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Lactate dehydrogenase leakage from cells was measured in cell supernatants using a Lactate Dehydrogenase Activity Assay Kit (Cambridge Bioscience) [6 (link), 63 (link)]. Where indicated in Results, LDH leakage from cells was normalized to the cellular DNA in the control challenge.
To examine potassium leakage, 7.5 × 105 cells were seeded in 75 cm2 culture flasks in complete media for 24 h, before treatment with or without 2 mM glutamine for a further 24 h in serum-free media. Media were then discarded and cells washed three times with potassium-free choline buffer (129 mM choline-Cl, 0.8 mM MgCl2, 1.5 mM CaCl2, 5 mM citric acid, 5.6 mM glucose, 10 mM NH4Cl, 5 mM H3PO4, pH 7.4; all Sigma). Cells were then incubated in choline-buffer with control medium or pyolysin for 5 min at 37°C. Subsequently, cells were washed three times in ice-cold choline-buffer and lysed in 0.5% Triton X-100 (Sigma) in double-distilled water for 20 min at room temperature with gentle agitation. Potassium was measured in the cleared lysates using a Jenway PFP7 flame photometer (Cole-Parmer, Stone, Staffordshire, UK).
To examine potassium leakage, 7.5 × 105 cells were seeded in 75 cm2 culture flasks in complete media for 24 h, before treatment with or without 2 mM glutamine for a further 24 h in serum-free media. Media were then discarded and cells washed three times with potassium-free choline buffer (129 mM choline-Cl, 0.8 mM MgCl2, 1.5 mM CaCl2, 5 mM citric acid, 5.6 mM glucose, 10 mM NH4Cl, 5 mM H3PO4, pH 7.4; all Sigma). Cells were then incubated in choline-buffer with control medium or pyolysin for 5 min at 37°C. Subsequently, cells were washed three times in ice-cold choline-buffer and lysed in 0.5% Triton X-100 (Sigma) in double-distilled water for 20 min at room temperature with gentle agitation. Potassium was measured in the cleared lysates using a Jenway PFP7 flame photometer (Cole-Parmer, Stone, Staffordshire, UK).
6
Quantifying Potassium Leakage in Cells
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To quantify the leakage of potassium cells were seeded in complete medium at 1.5 × 105 cells per well in 6-well culture plates and incubated for 24 h, before treatment with vehicle, 10 µM dexamethasone or 10 µM hydrocortisone in serum-free medium for 24 h. Supernatants were discarded, and the cells were washed 3 times with potassium-free choline buffer (129 mM choline-Cl, 0.8 mM MgCl2, 1.5 mM CaCl2, 5 mM citric acid, 5.6 mM glucose, 10 mM NH4Cl, 5 mM H3PO4, pH 7.4; all Merck), and then challenged with choline buffer, pyolysin or streptolysin O for 5 min, or with 8 µg/5 × 104 cells α-hemolysin for 15 min (no potassium leakage was detectable from HeLa cells after 5 min of α-hemolysin challenge). Cells were then washed 3 times in ice-cold choline buffer and lysed in 0.5% Triton X-100 (Merck) in distilled water for 20 min on a rocker at room temperature. Potassium was measured in supernatants and lysates using a Jenway PFP7 flame photometer (Cole-Parmer, Stone, Staffordshire, UK). The inter- and intra-assay coefficients of variation were < 4%.
7
Mineral Content Analysis Protocol
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The amount of minerals present in the sample was determined as described by AOAC
19 . The ash of the sample obtained was dissolved in 10ml of 2M HNO
3 and boiled for 5 min, filtered through Whatman No.1 filter paper into volumetric flask. The filtrate was made up with distilled water to 50 ml and used for determination of minerals content. Zinc (Zn), Manganese (Mn), Magnesium (Mg) and Iron (Fe) were determined by using Atomic Absorption Spectrophotometer (AAS 220GF, Buck). The standard curve for each mineral was prepared from known concentrations of mineral and the mineral content of the samples was estimated from the standard curve, while sodium and potassium content were determined using Jenway flame photometer PFP7 (Cole-palmer, UK).
19 . The ash of the sample obtained was dissolved in 10ml of 2M HNO
3 and boiled for 5 min, filtered through Whatman No.1 filter paper into volumetric flask. The filtrate was made up with distilled water to 50 ml and used for determination of minerals content. Zinc (Zn), Manganese (Mn), Magnesium (Mg) and Iron (Fe) were determined by using Atomic Absorption Spectrophotometer (AAS 220GF, Buck). The standard curve for each mineral was prepared from known concentrations of mineral and the mineral content of the samples was estimated from the standard curve, while sodium and potassium content were determined using Jenway flame photometer PFP7 (Cole-palmer, UK).
8
Mineral Content Determination Protocol
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The amount of minerals present in the sample was determined as described by AOAC (2010) . The ash of the sample obtained was dissolved in 10 mL of 2 M HNO3 and boiled for 5 min, and filtered through a Whatman no.1 filter paper into a volumetric flask. The filtrate was made up with distilled water to 50 mL and used for determination of mineral content. Calcium, phosphorus, and magnesium were determined by atomic absorption spectrophotometer (Model 220 GF, Buck Scientific, East Norwalk, CT, USA). The standard curve for each mineral was prepared from known concentrations of mineral and the mineral content of the samples was estimated from the standard curve while sodium and potassium content were determined using Jenway Flame Photometer PFP7 (Cole- Parmer Instrument Co., Ltd., Eaton Socon, UK) (AOAC, 2010 ).
9
Mineral Content Analysis of Ash Samples
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Ash from the samples was liquefied in 10 mL of 2 M HNO3, boiled for 5 min, and filtered into volumetric flask. The filtrate was made up with distilled water to 50 mL. The concentrations of magnesium, manganese, iron, and zinc were determined using atomic absorption spectrophotometer (220GF, Buck Scientific Inc., Norwalk, CT, USA). Standard curves for each mineral were prepared from known concentrations of minerals (AOAC, 2010 ). Sodium content was determined using Jenway flame photometer PFP7 (Cole-Parmer Instrument Co., Ltd., Cambridgeshire, UK).
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