All animal experiments were performed in compliance with the guidelines established by the USC Institutional Animal Care and Use Committee. Rats were administered doses of 10 nanomoles of rhodamine covalently linked to the amino terminus of recombinant L4F-A192. Rats were administered fluorescein L4F, at doses up to 50 nanomoles. L4F was chemically synthesized as follows, Met-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Phe-Leu-FITC (LifeTein, South Plainfield, NJ). Rats were bled at indicated time points from the tail into plasma separator tubes with lithium heparin (Becton, Dickinson and Co, Franklin Lakes, NJ). Serum was collected by spinning down blood samples and collecting supernatant. Plasma concentrations were determined using a calibrated fluorescence microplate assay run on a BioTek Synergy H1 Hybrid Multi Mode Microplate Reader (Biotek, Winooski, VT).
Biotek synergy h1 hybrid multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader is a versatile instrument designed for various microplate-based assays. It combines absorbance, fluorescence, and luminescence detection capabilities in a single system.
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Lab products found in correlation
Lithium heparin, by BD (1 mentions)
Fastdna spin kit, by MP Biomedicals (1 mentions)
Complex 1 enzyme activity microplate assay kit, by Abcam (1 mentions)
Mitotox complex 2 3 oxphos activity assay kit, by Abcam (1 mentions)
Complex 2 enzyme activity microplate assay kit, by Abcam (1 mentions)
Complex 4 rodent enzyme activity microplate assay kit, by Abcam (1 mentions)
6 protocols using biotek synergy h1 hybrid multi mode microplate reader
1
Rat Fluorescence Assay for L4F Peptide
2
Soil DNA Extraction for qRT-PCR
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The total genomic DNA was extracted from 0.5 g newly collected soil with three replicates using the Fast DNA™ Spin Kit (MP Biomedicals, LLC, Santa Ana, USA) according to the manufacturer’s instructions. DNA concentration and quality were measured by calculating their absorbance (A260 and 280 nm) using BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, USA). DNA was diluted with sterile water to a final concentration of 20 ng μL− 1 for qRT-PCR. The integrity of the DNA extracts was ensured by electrophoresis and was stored at − 80 °C awaiting sequencing.
3
Fluorescence-based CYP1B1 Activity Assay
4
Multiprotein Complex Activity Assays
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Mitochondrial respiratory chain complex I II, III, and IV were measured by Complex I Enzyme Activity Microplate Assay Kit (ab109721, abcam, Cambridge, United Kingdom), Complex II Enzyme Activity Microplate Assay Kit (ab109908, abcam, Cambridge, United Kingdom), MitoTox Complex II + III OxPhos Activity Assay Kit (ab109905, abcam, Cambridge, United Kingdom), and Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911, abcam, Cambridge, United Kingdom) respectively according to the manufacturer’s recommendation. Briefly 80 μg mitochondrial proteins were loaded in a 96-well plate, and the enzyme activity was determined colorimetrically with BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, United States) by detecting the light absorption value of the sample at 450 nm (complex I), 600 nm (complex II), and 550 nm (complex III and complex IV) respectively.
5
Long-term Cellular Proliferation Assay
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H460- and HCC827-pcDNA control and H460- and HCC827-DUSP10 overexpression cells were seeded at a density of 0.08×106 cells/well in 6-well plates and incubated overnight at 37°C with 5% CO2. Subsequently, cells were incubated in serum free RPMI for 20 h at the same condition. After serum starvation, serum free RPMI was replaced with a complete RPMI for assessment of basal proliferation over a period of 5 days. At each time point, RPMI was removed and 500 μl crystal violet stain (0.5% crystal violet in 20% methanol) was added into the wells and was shaken for 5 min. Crytal violet stain was then removed, cells were washed with RO water and the plates were left to air-dry for 15 min. After drying, the stain was solubilized in 500 μl solubilisation buffer (0.1M sodium citrate in 50% absolute ethanol). Analysis of relative proliferation rate was done by measuring absorbance at 545nm using BioTek Synergy™ H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA).
6
Minimal Inhibitory Concentration Assay for rtVWF
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The minimal inhibitory concentration (MIC) for rtVWF was evaluated using the broth microdilution assay. Briefly, the bacterial cultures were diluted to a final O.D. 600 nm of 0.001 in respective broth. One hundred microliters of the diluted bacterial cultures were added to each well, of a 96-well plate, in triplicate. rtVWF was diluted in sterile molecular grade water to 500, 250, 125, 62.5 and 31.25 μM. Then 11 μl of each solution were added to wells in triplicate, resulting in a final concentration of rtVWF at 45.4, 22.7, 11.3, 5.7 and 2.8 μM. Some wells received bacteria alone with 11 μl of sterile water, which served as a vehicle control, or 11 μl of kanamycin sulfate (45.4 mg/ml). Some wells also received 100 μl of broth and 11 μl of sterile water which served as a background control. All 96-well plates were sealed with parafilm and incubated at either 14 • C for 3 days (F. psychrophilum) or 28 • C for 7 h (Y. ruckeri and S. iniae). This panel of important fish bacterial pathogens was selected due to differences in Gram-negative and Gram-positive properties. The hourly growth of bacteria was monitored using a spectrophotometer at an O.D. of 600 nm (BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader, BioTek Instruments). To establish growth curves, the background values were averaged and subtracted from each reading.
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