To determine the effect of ATP (Sigma) and/ or IL‐33 (50 ng/ml) (PeproTech) on the intracellular Ca2+ concentration [Ca2+]I, 5x106 BMMCs were resuspended in KREPS‐HEPES buffer (20 mM HEPES pH 7·4,135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0·4 mM KH2PO4 and 5·5 mM glucose) and incubated with 0·2 µM Fura‐2/AM (Thermo Fisher Scientific, Waltham, MA) for 30min (37°celsius, 5% CO2). After centrifugation, cells were recovered in KREBS‐HEPES buffer supplemented with 1 mM CaCl2 and diluted to a final density of 1 × 106 cells/ml. Cells were transferred onto black 96‐well microplates PS F‐bottom (Greiner bio‐one), and the signal was monitored by a NOVOstar microplate reader (BMG Labtechnologies GmbH) at 37°C : emission at 510 nm, excitation at 340 nm (Ca2+‐bound Fura‐2) and 380 nm (free Fura‐2). By subsequent cell lysis with triton X‐100, the maximal fluorescence signals were determined, followed by chelating Ca2+ with 20 mM EDTA to assess the minimal fluorescence.
F bottom
Manufactured by Greiner
Sourced in Germany
The F-bottom is a laboratory equipment product designed for laboratory applications. It serves as a stable and secure platform for various laboratory tasks and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Il 33, by Thermo Fisher Scientific (1 mentions)
Novostar microplate reader, by BMG Labtech (1 mentions)
Fluostar omega plate reader, by BMG Labtech (1 mentions)
Multiskan go, by Thermo Fisher Scientific (1 mentions)
96 well 0.45 μm pore filter plate, by Pall Corporation (1 mentions)
5 protocols using f bottom
1
Intracellular Calcium Measurement in BMMCs
2
Quantifying Oral Protease Activity
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To quantify the protease activity in the oral rinses, one ml aliquots from the first rinses of each participant were retrieved from − 80 °C storage and thawed on ice. The protease activity was determined using black 96-wells microplates (F Bottom, Greiner Bio- One GmbH, Frickenhausen, Germany). Each microwell was filled with 70 ml of PBS, and 8 μM protease substrate PEK-054 ([FITC]-NleKKKKVLPIQLNAATDK-[KDbc]), a substrate for total protease activity [21 (link)]. As a positive control, trypsin from bovine pancreas was added in duplicate in two-fold serial dilutions, and sterile PBS was used as a negative control. Samples from periodontitis patients and controls were defrosted and 30 μl was added to each microwell. The increase in fluorescence was monitored over 60 min using a fluorescence microplate reader (Fluostar Galaxy, BMG Laboratories, Offenburg, Germany) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Relative fluorescence (RF) values were obtained for periodontitis patients and controls. The total protease activity was defined in RF per Unit (RF U).
3
High Pressure Protein Release Assay
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For the determination of protein release upon high pressure treatment, an aliquot of 500 μL of a pressure-treated sample (108–109 cfu mL− 1) was transferred to a sterile reaction tube and centrifuged (2800×g, 5 °C, 15 min). The supernatant was filter-sterilized (0,2 μM pore size, Sarstedt, Nürnbrecht, Germany) and protein concentration was measured on black 96-well microtiter plates (F-bottom, Greiner bio-one, Frickenhausen, Germany) using the Pierce™ Coomassie Plus (Bradford) assay kit (ThermoFisher, Waltham, MA, USA) according to the micro MTP protocol provided by the manufacturer. The samples were incubated at 25 °C in the dark for 10 min and absorbance at 595 nm was measured in a FLUOStar Omega plate reader (BMG LABTECH GmbH, Ortenberg, Germany). Bovine serum albumin provided with the kit was used to establish a standard curve for protein concentrations between 0 and 25 μg mL− 1.
4
Seed Protein Extraction and Quantification
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Total protein was extracted from homogenized seeds samples of each accession using an extraction buffer (70 mM Tris HCl, 25 mM KCl, 1 mM MgCl2, five mM EDTA, 5% glycerol, 0.1% Triton X-100, and 15 mM β-mercaptoethanol). The extracts were filtered using a 96-well 0.45-μm-pore filter plate (Pall Life Sciences, New York, USA), and filtrates were used to determine protein content with the Bradford Protein Assay Kit (AMRESCO Inc., Solon, OH). Extracts of 20 µl were incubated with 180 µl buffer for 2 min in a 96-well microplate (F-bottom, Greiner Bio-One, Kremsmünster, Austria) before measuring the absorbance at 595 nm in a spectrophotometer (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Before measuring samples, 0.5 mg/mL BSA solution was used to prepare a standard curve to detect protein concentrations, and the total protein was reported as µg per mg of seed.
Statistical procedures such as ANOVA, Student t-Test, and the Principal component analysis (PCA) were performed using JMPR 15.2.0 (SAS Institute Inc., Cary, NC, USA) statistical package.
Statistical procedures such as ANOVA, Student t-Test, and the Principal component analysis (PCA) were performed using JMPR 15.2.0 (SAS Institute Inc., Cary, NC, USA) statistical package.
5
Hydrogel-Based Contraction Assay for hDPSCs
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Gels were prepared as described above with some modifications. Donor-matched hDPSCs or d-hDPSCs were added to the hydrogel mixture in a final seeding density of 10 6 cells/ml. Cellseeded collagen hydrogels were cast in duplicate in a 96-well plate (Greiner F-bottom; 75 μl/well) for six different time points (0, 2, 4, 6, 8 and 24 hours). After 10 min of setting at 37 °C, gels were immersed in 200 μl standard culture medium and detached from the wells using a fine spatula. Four independent experiments, i.e. cells from four different donors (n = 4), were carried out and for each experiment, free-floating hydrogel contraction was assessed both macro-and microscopically at the indicated time points. For macroscopic analysis, the medium was removed and digital images were taken. ImageJ software was used to determine the area of the upper surface of the gels. Collagen gel size was defined as a percentage of the initial hydrogel area. For microscopic analysis, duplicate gels were transferred to µ-Slide 8 Well ibiTreat (Proxylab, Beloeil, Belgium) and 3 random regions without cells in the field of view were imaged in the centre of each hydrogel. For one donor (n = 1), also 3 random regions at the edge of each hydrogel were imaged.
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