To obtain release profiles of IL-4, ALN and Ca2+, the PEEK-IL4 samples were placed in a 24-well plate containing 1 ml PBS per well and incubated at 37 °C. At prescribed time points, supernatant was collected and refilled with fresh PBS. The released IL-4 was quantified by the enzyme-linked immunosorbent assay (ELISA) kit (Valukine, R&D Systems, USA) following the manufacturer's instructions. The concentration of released ALN was determined by UV–visible spectrophotometry (UV-US, TU-1810, Pulse, China) at 293 nm according to the previously reported method [28 ]. The Ca2+ concentration was measured using a calcium colorimetric assay kit (Beyotime, China) according to the manufacturer's instructions.
Calcium colorimetric assay kit
Manufactured by Beyotime
Sourced in China
The Calcium Colorimetric Assay Kit is a laboratory equipment product that measures the concentration of calcium in a sample. It utilizes a colorimetric reaction to quantify the amount of calcium present, providing a reliable and accurate method for calcium analysis.
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Alkaline phosphatase assay kit, by Beyotime (1 mentions)
Cell lysis buffer for western and ip kit, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
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Ethanol c2h6o, by Sinopharm (1 mentions)
Lsm 900 confocal microscope, by Zeiss (1 mentions)
Multiskan fc microplate reader, by Thermo Fisher Scientific (1 mentions)
33 protocols using calcium colorimetric assay kit
1
Quantifying IL-4, ALN, and Ca2+ Release
2
Calcium Content Colorimetric Assay
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Ca2+ content (mmol·L−1) was measured using a calcium colorimetric assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) [48 (link)].
3
Quantifying Cellular Alkaline Phosphatase and Calcium
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The cells were lysed by cell lysis buffer for western and IP kit (Beyotime, China) following the manual of the manufacturers. Alkaline phosphatase assay kit (Beyotime, China) was used to detect the ALP activity in cell lysates. Samples and standards were added in 96-well plates, respectively. Then, para-nitrophenyl phosphate solution and ALP enzyme solution was added in sample and standard wells respectively and incubated for 10 min at 37 °C. Stop solution utilized to terminate the reaction. The 96-well plates analyzed with a microplate spectrophotometer (SpectraMax M2e, Molecular Devices, USA) at 405 nm.
The calcium in cells was detected by calcium colorimetric assay kit (Beyotime, China). The cells were lysed by sample lysis solution in the kit. Following centrifugation at 4 °C (12,000×g, 5 min), the supernatant was used for detecting. Fifty microliters standard and sample were added in 96-wells plates following the manual of manufactures. Test solution (test buffer to o-cresolphthalein complexone = 1:1) was added to each well (150 μl per well) and incubated for 10 min at room temperature in the dark. The 96-well plates analyzed with a microplate spectrophotometer (SpectraMax M2e, Molecular Devices, USA) at 575 nm.
The calcium in cells was detected by calcium colorimetric assay kit (Beyotime, China). The cells were lysed by sample lysis solution in the kit. Following centrifugation at 4 °C (12,000×g, 5 min), the supernatant was used for detecting. Fifty microliters standard and sample were added in 96-wells plates following the manual of manufactures. Test solution (test buffer to o-cresolphthalein complexone = 1:1) was added to each well (150 μl per well) and incubated for 10 min at room temperature in the dark. The 96-well plates analyzed with a microplate spectrophotometer (SpectraMax M2e, Molecular Devices, USA) at 575 nm.
4
Quantifying Calcium Levels in VZV-Infected Cells
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According to the manufacturer’s calcium colorimetric assay kit (# S1063S, Beyotime Biotechnology, China) operating manual, we determined the calcium ion content of the VZV-infected ARPE-19 and SH-SY5Y cell lysate and culture supernatant. After VZV infects ARPE-19 and SH-SY5Y cells, the cell culture medium is collected and rinsed with pre-cooled 1 × PBS two to three times. Add 200 μl of pre-cooled sample lysate to each sample to make the sample lysate fully contact the cells. After the cells are fully lysed, collect the cell lysate at 4°C, centrifuge at 12,000 × g for 5 min, and aspirate the supernatant. The standard curve of calcium ion content was obtained by calcium standard solution and chromogenic solution, and the absorbance value of each sample was quantified as the corresponding calcium ion concentration.
5
Calcium Concentration Measurement in Osteoclasts
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According to manufacturer's instructions, a calcium colorimetric assay kit (Beyotime Biotechnology) was used to measure calcium concentration. Briefly, the Raw264.7 cells were cultivated in 6-well plate with RANKL and RR (1.5 μM) stimulation for 24 or 48 hours and then washed with PBS and lysed with lysis buffer. Supernatants, collected after centrifugation, and working color solution were added, and then, the mixtures were incubated at RT for 10 mins to detect the absorbance at 575 nm through a microplate reader (MD Spectra Max i3x). Protein concentrations, used to normalize the amount of calcium, were measured through an BCA protein assay kit (Beyotime Biotechnology).
6
Pluronic F127 and Dopamine Hydrogel Synthesis
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Triblock poly (ethylene oxide)-bpoly(propylene oxide)-b-poly(ethylene oxide) Pluronic F127 (EO106PO70EO106, Mav = 12,600) and dopamine hydrochloride (C8H11NO2·HCl, DA·HCl) were brought from Sigma-Aldrich. Ethanol (C2H6O) was purchased from Sinopharm Chemical Reagent Co., Ltd. 1,3,5-trimethylbenzene (C9H12), Calcium chloride, hydrogen peroxide (H2O2, 30 wt% solution in water), ammonia water (25–28%), N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES), Hyaluronic acid sodium salt from Streptococcus equi, Rhodamine B (RhB) and dichloride [Ru(dpp)3]Cl2 were purchased from Aladdin (Shanghai, China). The lactate assay kit was brought from Nanjing Jiancheng Bioengineering Institute. Cell Counting Kit-8 (CCK-8), the Annexin V-FITC Apoptosis Detection Kit, and the Calcium Colorimetric Assay Kit were obtained from Beyotime Biotechnology Co., Ltd. Anti-CD16/32, anti-CD45-PE, anti-CD11b-APC, anti-CD11c-APC anti-F4/80-PE/Cy7, anti-CD80-FITC, anti-CD206-PE, anti-CD86-PE, anti-MHCII-V450, anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE/Cy7 were purchased from BioLegend. ELISA kits were purchased from Dakewei Biotech Co., Ltd.
All chemicals were of analytical grade and used without further purification. Deionized water (18.2 MΩ·cm resistivity at 25 °C) was used for all experiments.
All chemicals were of analytical grade and used without further purification. Deionized water (18.2 MΩ·cm resistivity at 25 °C) was used for all experiments.
7
Quantifying Shrimp Gill Calcium Content
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The shrimp were maintained in normal seawater or transferred to seawater containing 5 mg/L TAN. After transfer, the cell-permeating calcium chelator BAPTA-AM (Selleck; S7534) was injected into the shrimp hemocoel. The calcium content of shrimp gills was determined 30 min later. For the fluorescence probe assay, gills were collected and incubated with 3 µM of the calcium fluorescence probe, Fluo-3AM (Solarbio; F8840) for 30 min at 37°C. After washing with HEPES-buffered saline, gills were placed on slides containing glycerol. The slides were observed and imaged using an LSM 900 confocal microscope (Zeiss).
Calcium concentration was determined using a Calcium Colorimetric Assay Kit (Beyotime; S1063S), according to the manufacturer’s instructions. Briefly, gills were lysed using a lysate buffer. The lysate was centrifuged at 12,000 × g for 10 min at 4°C. The resulting supernatant was collected and incubated with the detection reagent at a ratio of 1:3. The incubation was continued for 10 min in the dark. The optical density at 575 nm was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific).
Calcium concentration was determined using a Calcium Colorimetric Assay Kit (Beyotime; S1063S), according to the manufacturer’s instructions. Briefly, gills were lysed using a lysate buffer. The lysate was centrifuged at 12,000 × g for 10 min at 4°C. The resulting supernatant was collected and incubated with the detection reagent at a ratio of 1:3. The incubation was continued for 10 min in the dark. The optical density at 575 nm was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific).
8
Quantifying Calcium Flux and Oxidative Stress in Leaves
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Leaves were used to detect Ca2+ flux using Non‐invasive Micro‐test Technology (NMT) as described previously.[55 (link)
] The Ca2+ content (mg·g−1) was measured using a calcium colorimetric assay kit (Beyotime Biotechnology Co., Ltd. Shanghai, China).
Leaves were incubated in 1 mg ml−1, pH 3.8, DAB‐HCl (Sigma‐Aldrich, USA) in the dark for 8 h. The cotyledons were then cleared by boiling in alcoholic lactophenol (95% ethanol: lactophenol, 2:1 v/v) for 20 min. The reddish color of the cotyledons was used as evidence of H2O2 and visualized using a Nikon D40 camera (Japan). The quantification of H2O2 was conducted using the test kit (G0112W) from Suzhou Grace Biotechnology Co., Ltd (Suzhou, China), following the manufacturer instructions. For SA extraction, plant tissues were ground into fine powder by liquid nitrogen, and 0.1 g was used from each sample following the method of.[26 (link)
] Samples were then analyzed using LC/MS.
] The Ca2+ content (mg·g−1) was measured using a calcium colorimetric assay kit (Beyotime Biotechnology Co., Ltd. Shanghai, China).
Leaves were incubated in 1 mg ml−1, pH 3.8, DAB‐HCl (Sigma‐Aldrich, USA) in the dark for 8 h. The cotyledons were then cleared by boiling in alcoholic lactophenol (95% ethanol: lactophenol, 2:1 v/v) for 20 min. The reddish color of the cotyledons was used as evidence of H2O2 and visualized using a Nikon D40 camera (Japan). The quantification of H2O2 was conducted using the test kit (G0112W) from Suzhou Grace Biotechnology Co., Ltd (Suzhou, China), following the manufacturer instructions. For SA extraction, plant tissues were ground into fine powder by liquid nitrogen, and 0.1 g was used from each sample following the method of.[26 (link)
] Samples were then analyzed using LC/MS.
9
Quantifying Nitric Oxide and Calcium in Leaves
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The concentration on NO in leaves was assayed by Griess reagent following a method provided by Zhang et al. [34 (link)]. Absorbency was determined with a spectrophotometer (540 nm). Through matching with the specification curve based on NaNO2 as standard, the amount of NO was assayed. The concentration of Ca2+ (mg⋅g− 1) was assayed with a calcium colorimetric assay kit (Beyotime Biotechnology Co, Ltd. Shanghai, China).
10
Calcium Homeostasis in WSSV-Infected Shrimp
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To determine whether WSSV infection affects the concentration of calcium ions, we used the intestine tissue of shrimp to detect the free calcium ions using a Calcium Colorimetric Assay Kit (Beyotime Biotechnology, Shanghai, China). Shrimp intestines were collected at 0, 10, 20, and 30 min, and 1, 2, 6, and 12 h post WSSV challenge for calcium detection. The intestine tissue (30 mg) was homogenized in 300 μL of sample lysis buffer using a glass homogenizer, followed by centrifugation at 14,000 × g for 5 min. First, the protein concentration of the supernatant was measured using the Bradford method. The supernatant was placed on ice for the subsequent measurement. Next, the calcium standard solution was diluted to different concentrations to create a standard curve. The concentration of the supernatant was detected following manufacture’ protocols using a microplate reader (TECAN M200 PRO, Tecan Group Ltd., Männedorf, Switzerland) at 575 nm.
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