Changes in intracellular sulfane sulfur levels were monitored with the SSip-1 DA probe (Takano et al., 2017 ). Cells were seeded in black flat-bottomed 96-well imaging plate (BD Falcon 353219) at a density of 166 x 103 cells/ml in DMEM. The following day the medium was discarded, cells washed with HBSS and loaded with SSip-1 DA in DMEM at a concentration of 5 μM for 1 h at 37°C. The cells were then washed twice with HBSS and the SSip-1 DA signal acquisition in HBSS was carried out by recording the fluorescence λex= 470 nm and λem= 520 nm. Experiments were performed in biological triplicates, using technical quadruplicates.
Falcon 353219
Manufactured by BD
The BD Falcon 353219 is a polystyrene tissue culture plate designed for cell culture applications. It features a flat bottom and clear surface, and is available in a range of well configurations to suit different experimental needs.
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Lab products found in correlation
Mitopy1, by Bio-Techne (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Bcecf am, by Thermo Fisher Scientific (1 mentions)
Synergy htx multi mode reader, by Agilent Technologies (1 mentions)
Clariostar fluorescence plate reader, by BMG Labtech (1 mentions)
Micro bio spin column, by Bio-Rad (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
6 protocols using falcon 353219
1
Monitoring Intracellular Sulfane Sulfur
2
H2S Quantification using HSip-1 DA Probe
3
Mitochondrial H2O2 Quantification Using MitoPY1
4
Intracellular pH Measurement in NLRP3 Knockout Cells
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NLRP3 knockout THP-1 cells were seeded at 50,000 cells per well and BMDM were seeded at 50,000 cells per well in black-walled clear bottom 96-well plate (BD Falcon 353219). Cells were preloaded with 5 μM BCECF-AM (Thermo, B1170) in HBSS (Thermo, 14025-092) for 30 min at 37 °C. Cells were then treated with activators in HBSS in 100 μL per well volume and pH values were determined using calibration wells containing 15 μM nigericin in calibration buffer (135 mM KCl, 2 mM K2HPO4, 20 mM HEPES, 1.2 mM CaCl2, 0.8 mM MgSO4) adjusted to pH of 5.5, 6.5, 7.5, and 8.5. For intracellular pH measurement under different extracellular pH conditions, “pH adjusted buffers” described above were used instead of HBSS. For pH measurement under glucose or galactose as carbon source, we used “pH adjusted buffers” at pH 7.9 without glucose, supplemented with either 10 mM glucose or 10 mM galactose. The fluorescence signal (Ex 485 nm/Em 528 nm) and background (Ex 380 nm/Em 528 nm) were measured using a Synergy HTX multi-mode reader (BioTek) set to 37 °C temperature. Background was subtracted from signal and a calibration curve was used to calculate intracellular pH.
5
Yeast Peroxiredoxin Redox Sensing
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Codon-optimized LicPRX1 and LicPRX2 (GenScript Biotech, Netherlands) were cloned into the EcoRI/HindIII restriction sites of p416TEF-roGFP2 [44 (link)] to yield p416TEF-roGFP2-LicPRX1 and p416TEF-roGFP2-LicPRX2. The remaining mutein-carrying plasmids were generated from wild type constructs following standard site-directed mutagenesis protocols. Constructs were transferred to BY4742 Δtsa1Δtsa2 S. cerevisiae strain [45 (link)]. Yeast genetically encoding roGFP2-LicPRX1/2 probes were employed in roGFP2 measurements, as described before [46 (link)]. Briefly, yeast cultures were grown to late exponential phase (OD600nm = 3–4), suspended in buffer 100 mM MES-Tris pH 6.0 to a final concentration of 7.5 OD600nm units ml−1. Aliquots of 180 μl of the yeast suspension were transferred to a flat-bottomed 96-well imaging plate (BD Falcon 353219), challenged with either 20 mM diamide (fully oxidized control), 100 mM DTT (fully reduced control) or H2O2 (0–1000 μM), and monitored for 90 min at 30 °C in CLARIOstar fluorescence plate reader (BMG Labtech). Oxidized and reduced roGFP2 were excited at 400 nm and 480 nm, respectively, and emission of both redox states measured at 520 nm. At each time point, the degree of roGFP2 oxidation (OxD) was calculated as before [46 (link)].
6
Ratiometric Redox Biosensor Calibration
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The purified Mrx1-roGFP2 protein was reduced with 10 mM dithiothreitol (DTT) for 20 min, desalted with Micro-Bio spin columns (Bio-Rad), and diluted to a final concentration of 1 µM in 100 mM potassium phosphate buffer, pH 7.0. The oxidation degree (OxD) of the biosensor was determined by calibration to fully reduced and oxidized probes which were generated by treatment of the probes with 10 mM DTT and 5 mM diamide for 5 min, respectively [42] (link). The thiol disulfides and oxidants were injected into the microplate wells (BD Falcon 353219) 60 s after the start of measurements. Emission was measured at 510 nm after excitation at 400 and 488 nm using the CLARIOstar microplate reader (BMG Labtech) with the Control software version 5.20 R5. Gain setting was adjusted for each excitation maximum. The data were analyzed using the MARS software version 3.10 and exported to Excel. Each in vitro measurement was performed in triplicate.
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