Sapphire 2

Manufactured by Tecan

Sourced in Austria

The Sapphire II is a multi-mode microplate reader developed by Tecan. It is designed to perform a wide range of absorbance, luminescence, and fluorescence measurement applications in life science research and drug discovery.

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Lab products found in correlation

Cobas integra 400 plus, by Roche (2 mentions) 4000 qtrap, by Thermo Fisher Scientific (2 mentions) Bio one 96 well plate, by Greiner (2 mentions) Enzyme linked immunosorbent assay, by Thermo Fisher Scientific (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Lambda protein phosphatase, by New England Biolabs (1 mentions) Pdgfr, by Merck Group (1 mentions) 96 well half area plate, by Corning (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Gene cycler, by Bio-Rad (1 mentions) Cobas integra 400, by Roche (1 mentions) Difmup, by Thermo Fisher Scientific (1 mentions)

15 protocols using sapphire 2

Fluorescence Polarization Measurements

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Single photon (SP) data were collected in a plate reader set up for fluorescence polarization measurements (Tecan Sapphire 2). A G-factor for the instrument was calculated from 2μM fluorescein in water. Measurements were performed in 96 or 384 well plates.

Dubach J.M., Vinegoni C., Mazitschek R., Fumene Feruglio P., Cameron L.A, & Weissleder R. (2014). In vivo imaging of specific drug target binding at subcellular resolution. Nature communications, 5, 3946.

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Real-time Fluorescence Anisotropy Monitoring

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The oscillating fluorescence signals were captured by a photodiode; amplified 10-fold by an analog negative-feedback amplifier locked-in to the 1 kHz source signal; low-pass-filtered and amplified again by 30-fold; and converted to a digital signal by a pair of 12-bit analog-to-digital converters inside the MCU. The integrated real-time monitoring GUI displayed raw fluorescence readings from both parallel (I_parallel) and perpendicular (I_{perpendicular}) channels in real-time (every 0.1 s). The anisotropy (r) was computed according to the equation r = F·(I_parallel – I_{perpendicular})/(I_parallel + 2·I_{perpendicular}), where F was a scaling factor (F = 4259) to match CODA values with those measured by a plate reader (Sapphire 2, TECAN). Noise fluctuations arising from both raw and computed data streams were precisely measured as the rolling standard deviation of the most recent 40 samples, to confirm that the system's measurement was reliable (see Fig. S2 for the flow of signal processing).

Lee C.Y., Degani I., Cheong J., Lee J.H., Choi H.J., Cheon J, & Lee H. (2021). Fluorescence polarization system for rapid COVID-19 diagnosis. Biosensors & Bioelectronics, 178, 113049.

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Fluorescence Anisotropy Measurements

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Fluorescence anisotropy values were measured with the excitation and emission
wavelengths of 470 and 525 nm, respectively. Fluorescence anisotropy
(r) was calculated using the following equation:
r = (I_x –
I_y)·(I_x +
2I_y)^–1, where
I_x and I_y are emission
intensities when the emission polarizers are in parallel with and perpendicular to
the excitation polarizer, respectively. The LOD was estimated by setting the
threshold at 3× SD above the background signal of samples without bacteria.
For comparison with the benchtop equipment (Sapphire 2, Tecan), we measured
Δr = r −
r_FAM, where r_FAM is the
fluorescence anisotropy only in the presence of FAM-DNA (62.5 nM) and
r is the fluorescence anisotropy in the presence of FAM-DNA (62.5
nM) along with its template. We varied the template concentrations (10, 20, 30, 40,
50, and 60 nM) to produce different amounts of hybridized FAM-DNA.

Park K.S., Huang C.H., Lee K., Yoo Y.E., Castro C.M., Weissleder R, & Lee H. (2016). Rapid identification of health care–associated infections with an integrated fluorescence anisotropy system. Science Advances, 2(5), e1600300.

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Comprehensive Biomarker Assessment in Clinical Research

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Blood and urine samples were obtained in sterile containers at baseline and at study end. All blood samples were centrifuged at 3500 rpm for 7 minutes to extract serum and plasma samples, which were kept frozen at –20°C until laboratory testing at an ISO 15189:2007 accredited laboratory (SAMM 688) located at Center Of Pathology Diagnostics and Research Laboratories (CPDRL), UiTM Sungai Buloh. Fasting blood glucose (FBG) was analyzed using the hexokinase method, while total cholesterol (TC), triglyceride (TG) and high-density lipoprotein–cholesterol (HDL-C) were measured by enzymatic reference methods. FBG, TC, TG, HDL-C, lipoprotein(a)(Lp[a]), [18 ] and high-sensitivity C-reactive protein (hs-CRP) [19 ] were measured on an automated analyzer (Cobas Integra 400 PLUS, Roche Diagnostics, Germany). LDL-C was derived using the Friedewald equation. HbA1c were analyzed using the Biorad D10 machine (Bio-Rad Laboratories, Singapore). Serum intercellular adhesion molecule-1 (ICAM-1) [20 ] and endothelial nitric oxide synthase (eNOS) [21 ] concentrations were measured by enzyme-linked immunosorbent assay (eBioscience Bender MedSystems, Vienna Austria). All absorbance was read on a microplate reader (Tecan Sapphire II, Austria). In addition to these parameters, other biochemical safety parameters (eg, red blood cell count and hematocrit) were measured.

Zainordin N.A., Hatta S.F., Mohamed Shah F.Z., Rahman T.A., Ismail N., Ismail Z, & Abdul Ghani R. (2019). Effects of Dapagliflozin on Endothelial Dysfunction in Type 2 Diabetes With Established Ischemic Heart Disease (EDIFIED). Journal of the Endocrine Society, 4(1), bvz017.

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Fasting Biomarker Measurement Protocol

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Blood samples were collected in the morning following 10 to 12 h of fasting. All blood samples were centrifuged at 3500 rpm for 7 min to extract serum and plasma samples which were kept frozen at −20 °C until laboratory testing at an ISO 15189:2007 accredited laboratory (SAMM 688). Fasting plasma glucose (FPG) was analyzed using the hexokinase method, while TC, triglyceride (TG), and HDL-c were measured by enzymatic reference methods, and hsCRP concentration was measured by turbidimetric assay. FPG, TC, TG, HDL-c and hsCRP were measured on an automated analyzer (Cobas Integra 400 PLUS, Roche Diagnostics, Germany). LDL-c concentration was derived using the Friedewald equation [16 (link)]. These tests were accredited by an international accreditation body (MS ISO 15189:2007, SAMM No. 688). Serum IL-6, sICAM-1 and E-selectin concentrations were measured by enzyme linked immunosorbent assay (ELISA) (eBioscience Bender MedSystems, Vienna Austria). Serum ox-LDL concentration was measured using an ELISA kit (Mercodia, Sweden). MDA concentration was measured by a method adapted from [17 (link)]. All absorbance were read on a microplate reader (Tecan Sapphire II, Austria). F2-isoprostanes concentration was analyzed by liquid chromatography-tandem mass spectrometry method on the 4000 QTRAP (Applied Biosystem, Canada) following pretreatment of the samples using diethyl ether.

Rahman T., Hamzan N.S., Mokhsin A., Rahmat R., Ibrahim Z.O., Razali R., Thevarajah M, & Nawawi H. (2017). Enhanced status of inflammation and endothelial activation in subjects with familial hypercholesterolaemia and their related unaffected family members: a case control study. Lipids in Health and Disease, 16, 81.

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EphA4 Biosensor Phosphorylation Assay

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The EphA4 biosensors were expressed with an N-terminal 6 × His tag in Escherichia coli and purified by nickel chelation chromatography. The fluorescence emission ratio of ECFP/FRET (478 nm/526 nm) of the biosensors was measured at an excitation wavelength of 430 nm using a fluorescence plate reader (TECAN, Sapphire II) before and after the addition of 1 mM ATP and EphA4 (1 μg mL⁻¹, Sigma), c-Src (1 μg mL⁻¹, Upstate), or PDGFR (1 μg mL⁻¹, Sigma) kinases in kinase buffer (50 mM Tris·HCl, 100 mM NaCl, 10 mM MgCl₂, 2 mM DTT, pH 8), respectively. Lambda protein phosphatase (New England Biolabs) was added at a final concentration of 12 units/μL. Trypsin (Gibco) was used at a final concentration of 0.25%.

Pan Y., Lu S., Lei L., Lamberto I., Wang Y., Pasquale E.B, & Wang Y. (2019). Genetically Encoded FRET Biosensor for Visualizing EphA4 Activity in Different Compartments of the Plasma Membrane. ACS sensors, 4(2), 294-300.

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Bulk FRET Measurements for Heterodimer Formation

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For bulk FRET measurements to test for heterodimer formation indicative of subunit exchange, samples were prepared as Cy3-labeled, Cy5-labeled, “co-labeled” Cy3/Cy5-labeled protein that involved simultaneous labeling with both fluorophores, or “mixed” samples consisting of Cy3-labeled protein and Cy5-labeled protein added to the same well. Protein samples were studied at 1 μM in SEB. Samples were incubated at the indicated temperature and measured in a 96-well half area plate (Corning, Corning, NY) sealed with a film cover between experiments (Excel Scientific, Victorville, CA). Fluorescence intensity measurements were made using a TECAN Sapphire II plate reader. Donor spectra were collected by exciting the samples at 540 nm and collecting emission spectra between 555–850 nm. Acceptor spectra were collected by exciting samples at 640 nm and collecting the emission spectra between 653–850 nm. The direct excitation of Cy5 was subtracted away from the donor spectra of the experimental sample using the donor spectra from a sample labeled with only Cy5 (eqn. 10) [41 (link)].
The corrected spectra were area normalized and the FRET efficiency, E, was calculated by donor quenching (eqn. 11):

E = 1 - \frac{I_{D A}}{I_{D}}

where I_DA is the fluorescence intensity of the donor (Cy3) in the presence of the acceptor (Cy5) and I_D is the fluorescence intensity of Cy3 in the absence of the Cy5.

Mersch K., Ozturk T.N., Park K., Lim H.H, & Robertson J.L. (2021). Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface. Journal of molecular biology, 433(8), 166886.

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RNA Extraction and cDNA Synthesis

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Cellular RNA was purified using the RNeasy^® Mini Kit (Qiagen Gmbh, Hilden, Germany) according to the manufacturer’s instructions. An optional on-column DNase digestion was also conducted (Qiagen cat. no. 79254). Flow-throughs were discarded by vacuum assisted aspiration and the procedure was conducted under laminar airflow. In the last step, the columns were transferred to new collection tubes (Biopure; Sigma Aldrich, St. Louis, MO, USA) and RNase free water (50 μL) was added directly to the membrane. The RNA was eluted by centrifugation for 1 min at 8000 g. The samples were frozen in −80° until further analyses. The RNA concentration in samples diluted in Tris-HCl (10 mM, pH 7.0) was determined spectrophotometrically at 260 nm using a Tecan Sapphire II fluorescence microplate reader (Tecan Gmbh, Salzburg, Austria). The purity of the samples was evaluated by measuring the absorbance ratio of 260/280 nm. Single-stranded cDNA was synthesized using a commercial kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The reverse transcription was performed using a thermal cycler (Bio-rad Gene cycler™, Hercules, CA, USA).

Scheers N, & Sandberg A.S. (2014). Iron Transport through Ferroportin Is Induced by Intracellular Ascorbate and Involves IRP2 and HIF2α. Nutrients, 6(1), 249-260.

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Biosensor Kinase Activity Assay

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Biosensor was expressed with N-terminal 6× His-tag in Escherichia coli and purified by nickel chelation chromatography as previous described.^{40 (link)} Fluorescence emission spectrum with 430 nm excitation of purified biosensor with a final concentration of 1 μM was measured in a 96-well plate using a fluorescence plate reader (TECAN, Sapphire II). Emission ratios of ECFP/FRET (478/526 nm) were measured in kinase buffer (50 mM Tris pH 8, 100 mM NaCl, 10 mM MgCl2, 2 mM dithiothreitol, 1 mM ATP) at 30°C before and after the addition of 1 μg/ml active ZAP-70 kinase (Calbiochem).

Li K., Xiang X., Sun J., He H.T., Wu J., Wang Y, & Zhu C. (2016). Imaging Spatiotemporal Activities of ZAP-70 in Live T Cells Using a FRET-based Biosensor. Annals of biomedical engineering, 44(12), 3510-3521.

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Biomarker Assessment for Cardiovascular Risk

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Blood samples were collected in the morning following 10 to 12 hours of fasting. All blood samples were centrifuged at 3500 rpm for 7 minutes to extract serum and plasma samples which were kept frozen at −20°C until laboratory testing. Fasting plasma glucose was analyzed using the hexokinase enzymatic reference method on an automated analyzer. TC, TG, and HDL-c were measured by enzymatic reference methods, while hsCRP concentration was measured by turbidimetric assay on an automated analyzer (Cobas Integra 400 by Roche, Germany). LDL-c concentration was calculated using the Friedewald equation. Serum IL-6, sVCAM-1, sICAM-1 and E-selectin concentrations were measured by enzyme linked immunosorbent assay (ELISA) (eBioscience Bender MedSystems, Vienna Austria). Serum ox-LDL concentration was measured using an ELISA kit (Mercodia, Sweden). MDA concentration was measured by a method adapted from Ledwozyw et al, (1986). All absorbances were read on a microplate reader (Tecan Sapphire II, Austria). F₂-isoprostanes concentration was analyzed by liquid chromatography-tandem mass spectrometry method on the 4000 QTRAP (Applied Biosystem, Canada) following pretreatment of the samples using diethyl ether.

Wan Ahmad W.N., Sakri F., Mokhsin A., Rahman T., Mohd Nasir N., Abdul-Razak S., Md Yasin M., Mohd Ismail A., Ismail Z, & Nawawi H. (2015). Low Serum High Density Lipoprotein Cholesterol Concentration is an Independent Predictor for Enhanced Inflammation and Endothelial Activation. PLoS ONE, 10(1), e0116867.

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