Purified RNAs were diluted to 1 μM in buffer containing 100 mM KCl, 1 mM MgCl2, 40 mM HEPES (pH 7.5). RNAs were then heated up to 75°C for 5 min and then cooled to room temperature. RNA solutions were then mixed with an equal volume of the same buffer containing 20 μM DFHBI-1T or DFHO (synthesized (Song et al., 2014 (link), 2017 (link)) or Lucerna 410-1mg). Samples contained a final concentration 0.5 μM RNA and 10 μM DFHBI-1T or DFHO, unless otherwise noted. After 1 h incubation, fluorescence signal of each sample was measured using a Fluoromax-4C (Horiba Scientific) with 460 nm excitation and 505 nm emission, 5 nm slit widths, and 0.1 s integration time. Background signal was detected with the RNA-free sample containing the same concentration of DFHBI-1T in 100 mM KCl, 1 mM MgCl2, 40 mM K-HEPES (pH 7.5). The detected background signal was subtracted from the signal obtained from each RNA-containing sample. Some fluorescence data was collected using a SpectraMax M-series plate reader (Molecular Devices) (figures 5 B and C , S1 B , C , F , and G , S3 , S4 , and S5 A and B ). Results are shown for n=3 biological replicates, unless otherwise stated. No blinding was used in these experiments.
Spectramax m series
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M series is a line of microplate readers manufactured by Molecular Devices. The core function of these instruments is to measure absorbance, fluorescence, and luminescence in microplates for various applications in life science research and drug discovery.
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7 protocols using spectramax m series
1
Fluorescent RNA Visualization Assay
2
Cryopreserved PBMC Cytokine Assay
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Example 40
Cryopreserved human PBMCs were obtained from AllCells (cat# PB003F). After thawing/dilution protocols using RPMI medium supplemented with 5% FBS (heat inactivated), 100 ul/well of 1×106 cells/ml were plated into 96 well tissue culture plates (Corning). Cells were then pre-incubated for 1 hr at 37° C. in humidified 5% CO2 and 95% air with test compounds diluted in DMSO (final DMSO concentration 0.3%). Each compound was tested at 10 concentrations in duplicate wells. After the pre-incubation, 100 ng/ml LPS (E. Coli; Sigma) in RPMI media with 5% FBS was added for a 6 hr incubation at 37° C. in humidified 5% CO2 and 95% air. Controls on each plate included cells and LPS only, cells and media only (no LPS), and media only. After the 6 hr incubation, plates were centrifuged and the supernatants transferred to a new plate and frozen for subsequent TNFalpha analysis. Human TNFalpha was analyzed by ELISA according to the manufacturer's instructions (BD Sciences, BD OptEIA™ Cat#550610) and analyzed on a SpectraMax M series (Molecular Devices) microplate reader at OD 450 nm. IC50s were calculated using XLFit4 curve fitting software (IDBS) and a 4-parameter one-site sigmoid dose response fit. IC50 data for compounds are shown in Table 6.
3
Quantification of SmNACE Enzymatic Activity
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The NAD glycohydrolase activity of rSmNACE [17 (link)] and native SmNACE was measured as previously described [38 (link)] using 1,N6-ethenonicotinamide adenine dinucleotide (ε-NAD; MilliporeSigma). Briefly, increasing concentrations of rSmNACE (10-100ng) or 1 parasite/well were incubated with 20μM ε-NAD. Hydrolysis of ε-NAD by SmNACE leads to formation of NAM and fluorescent ε-ADPR. The production of fluorescent ε-ADPR was measured (λexc = 310 nm and λem = 410 nM) at 37°C for 60min using a Spectramax M Series microplate reader (Molecular Devices). The amount of native, enzymatically active SmNACE expressed by male and female S. mansoni was calculated using a standard enzyme activity curve generated with known amounts of rSmNACE. A non-linear regression algorithm (GraphPad, Prism) was used to calculate the IC50 value of the SmNACE inhibitor CMP1 [38 (link)] following incubation of 2 male + 2 female worms in 200μl HBSS with increasing concentrations of CMP1.
4
ETC Activity Measurement of M. smegmatis IMVs
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The ETC activity was measured as previously described with minor modifications64 (link). Briefly, M. smegmatis IMVs were resuspended at 0.1125 mg/mL in 10 mM HEPES (pH 7.5) buffer. Twenty five µL of suspended IMVs were distributed in a clear-bottom, transparent 96-well plate (Corning), containing an equal volume of 4 mM 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Drug treatments were made by adding DMSO, four concentrations of 2B8 (62.5 µM to 125 µM), 125 µM RA13, 15 µM CCCP, 18 µM BDQ, 80 µM TRZ, and 5 mM potassium cyanide (KCN). ETC activity was initiated by adding 75 µL of 0.2% Triton X-100 in PBS supplemented with either 1 mM NADH, 130 mM sodium succinate or both. Absorbance at 490 nm was immediately monitored at 37 °C for 10 minutes using a SpectraMax M series multi-mode plate reader (Molecular Devices, Sunnyvale, CA, USA).
5
Serum Inflammatory Biomarkers in Monotherapy
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Venous blood samples (5 ml) were collected at the onset of tapering (first visit) from all monotherapy subjects enrolled in the study for serum biomarkers level estimation. The serum was separated by centrifugation and stored in a −80°C deep freezer till analysis. The inflammatory biomarkers (TNF-α, IL-1 β, IL-6, IL-10, and HMGB1) were estimated through enzyme-linked immunosorbent assay kits (R and D Systems, Minneapolis, MN 55413, USA). The kits were run as per the instruction manual from the manufacturer and absorbance was recorded through Multi-Mode Microplate Readers (SpectraMax® M Series, Molecular Devices, California, USA). For comparison purposes, blood samples of healthy control subjects (n = 52) were also collected, and the above inflammatory markers were estimated in them.
6
Quantifying Circulating Myokine Levels
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Plasma levels of the myokines oncostatin-M, secreted protein acidic and rich in cysteine (SPARC), irisin and interleukin-15 (IL-15) were measured by enzyme-linked immunosorbent assay (ELISA; LSBio, Shirley, MA, USA) according to the manufacturing protocol. Myokine concentrations were measured using SpectraMax M Series multimode microplate readers (Molecular Devices, San Jose, CA, USA) at 595 nm absorbance.
7
MTT Assay for Uterine Cell Viability
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Uterine epithelial cells were seeded at 2.5 × 104 cells/well in 24-well plates and incubated in the absence or presence of different concentrations of AnxA1, Boc-2, cyclosporine H, or WRW4 over either 24 or 48 h. Following the incubation period, the medium was carefully removed and 300 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/mL; #M5655, Sigma-Aldrich) was added in each well. Cells were maintained at 37 °C for 3 h, after which the supernatant was removed and 200 μL of dimethyl sulfoxide (DMSO; #276855, Sigma-Aldrich) was added into each well and homogenized for 15 min. Absorbance was determined using a spectrophotometer at 575 nm (SpectraMax M Series, Molecular Devices, San Jose, CA, USA). Results were expressed as the percentage of viable cells relative to NT cells (control).
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