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Saphire2 plate reader

Manufactured by Tecan
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Sourced in Switzerland

The Saphire2 plate reader is a multimode microplate reader designed for a variety of applications in life science research. It features high-performance monochromators for flexible wavelength selection and a wide detection range. The Saphire2 provides accurate and sensitive measurements for absorbance, fluorescence, and luminescence detection.

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Lab products found in correlation

Celltiter 96 non radioactive cell proliferation assay mtt, by Promega (1 mentions) Imap binding buffer a, by Molecular Devices (1 mentions)

5 protocols using saphire2 plate reader

1

CRISPR-Mediated Genetic Manipulation of Candida albicans

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C. albicans strain SC5314 was used for all experiments unless otherwise noted. The fluconazole-resistant C. albicans strain Can90 was provided by the Massachusetts General Hospital. Yeast strains were grown in YPD medium supplemented with 0.27 mM uridine and selected using Nat at a concentration of 200 μg/ml. Transformations were performed using the lithium acetate method (27 (link)). Flipout of NatR gene from Cas9-expressing Duet vector pV1025 was done by induction of flippase by growth in Difco yeast carbon base with bovine serum albumin, and screening for isolates that had lost the NatR gene. Filamentation experiments were performed with yeast grown overnight in liquid YPD, washed twice in RPMI 1640 medium (cat. #22400-105, Life Technologies) supplemented with 10% FBS, and incubated in RPMI + 10% FBS for the indicated time at a starting optical density (OD) of 0.1. Growth curves were performed in a clear-bottom 96-well plate and incubated with shaking at 30°C in a Tecan Saphire2 plate reader, reading OD at 600 nm every 5 min for the indicated time. YPD-grown overnight yeast cultures were used to inoculate these wells to an initial OD of 0.05. CRISPR-mutagenized loci were verified by sequence analysis of PCR products amplified from the target locus and by restriction digest where applicable.
Vyas V.K., Barrasa M.I, & Fink G.R. (2015). A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Science Advances, 1(3), e1500248.
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2

Cytotoxicity Assay for Cholesterol-Dependent Cytolysins

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HAP1 WT or single-gene knock-out cells were seeded in 96-well plates at 2 x 104 cells per well per 200 μl in at least 3 replicates. ILY, VLY, or PLY (CDC) serial dilutions in culture medium were added to the cells the next day. Cells were incubated with CDCs for 1h at 37°C. Subsequently, 20 μl of MTS reagent was added to each well. After incubating cells at 37°C for 4-6h, plates were scanned using Tecan Saphire 2 plate reader. Absorption at 490 nm was measured. Cells without toxin were used as a negative control, medium without cells–a positive control. Data were plotted and fitted with a non-linear regression model in GraphPad Prism 8. IC50 values were used to compare cell line resistances to ILY.
Drabavicius G, & Daelemans D. (2021). Intermedilysin cytolytic activity depends on heparan sulfates and membrane composition. PLoS Genetics, 17(2), e1009387.
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3

Genetic Manipulation and Phenotypic Analysis of C. albicans

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C. albicans strain SC5314 was used for all experiments unless otherwise noted. The fluconazole-resistant C. albicans strain Can90 was provided by the Massachusetts General Hospital. Yeast strains were grown in YPD medium supplemented with 0.27 mM uridine and selected using Nat at a concentration of 200 μg/ml. Transformations were performed using the lithium acetate method (27 (link)). Flipout of NatR gene from Cas9-expressing Duet vector pV1025 was done by induction of flippase by growth in Difco yeast carbon base with bovine serum albumin, and screening for isolates that had lost the NatR gene. Filamentation experiments were performed with yeast grown overnight in liquid YPD, washed twice in RPMI 1640 medium (cat. #22400-105, Life Technologies) supplemented with 10% FBS, and incubated in RPMI + 10% FBS for the indicated time at a starting optical density (OD) of 0.1. Growth curves were performed in a clear-bottom 96-well plate and incubated with shaking at 30°C in a Tecan Saphire2 plate reader, reading OD at 600 nm every 5 min for the indicated time. YPD-grown overnight yeast cultures were used to inoculate these wells to an initial OD of 0.05. CRISPR-mutagenized loci were verified by sequence analysis of PCR products amplified from the target locus and by restriction digest where applicable.
Vyas V.K., Barrasa M.I, & Fink G.R. (2015). A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Science Advances, 1(3), e1500248.
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4

MTT Assay for Cell Viability

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Measurement of cell viability upon compound treatment was performed using the CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) (Promega, Madison, WI, USA) according to the manufacturer’s instructions. In short, 1 × 104 MDCKII cells per well were seeded in 96-well plates. The cells were treated for 24 h with eight different inhibitor concentrations, diluted in an MDCKII cell culture medium. As a vehicle control, cells were treated with DMSO only. After incubation of the cells with the MTT-like substrate for 4 h, a stop solution was added, and the plates were further incubated at 37 °C overnight to allow solubilization of the blue crystals. Absorbance at 570 nm was measured on a Saphire2 plate reader (TECAN, Männedorf, Switzerland), and the cell viability was calculated in comparison to the DMSO control treatment.
Ganter B., Zickler M., Huchting J., Winkler M., Lüttjohann A., Meier C., Gabriel G, & Beck S. (2023). T-705-Derived Prodrugs Show High Antiviral Efficacies against a Broad Range of Influenza A Viruses with Synergistic Effects When Combined with Oseltamivir. Pharmaceutics, 15(6), 1732.
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5

Kinase Assays for MRCKα, MRCKβ, ROCK1, and ROCK2

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MRCKα, MRCKβ, ROCK1 and ROCK2 assays were performed as described (19 (link)). Recombinant kinase proteins (Life Technologies) at 8-12 nM were incubated at room temperature for 60 min with 100 nM FAM-S6-ribosomal protein derived peptide (Alta Biosciences) with 1 µM ATP and 0.5 mM MgCl2 in 20 mM Tris buffer (pH 7.4) containing 0.01% (v/v) Tween20, 1 mM DTT for MRCKα and β; or 1 μM ATP, 10 mM MgCl2 in 20 mM Tris buffer (pH 7.5) containing 0.25 mM EGTA, 0.01% (v/v) Triton X-100, 1 mM DTT for ROCK1 and ROCK2. Reactions were stopped by adding 2 volumes 0.25% (v/v) IMAP binding reagent in 1X IMAP binding buffer A (Molecular Devices). After 30 min to allow binding reagent to bind phosphorylated peptide, fluorescence polarization was measured on a Tecan Saphire2 plate reader at excitation (470 nm) and emission (530 nm) wavelengths. Inhibition was calculated using no inhibitor (0%) or no enzyme (100%) controls. MRCKα and MRCKβ kinases assays by out-sourced supplier were carried out in 50 mM HEPES pH 7.5, 0.01% (v/v) BRIJ-35, 10 mM MgCl2, 1 mM EGTA and 2 µM Ser/Thr 13 peptide substrate, with 1 hour incubation before addition of development reagent and quantification by fluorescence resonance energy transfer (FRET). Kinase selectivity profiling was performed by Invitrogen with indicated concentrations of BDP8900 or BDP9066.
Unbekandt M., Belshaw S., Bower J., Clarke M., Cordes J., Crighton D., Croft D.R., Drysdale M.J., Garnett M.J., Gill K., Gray C., Greenhalgh D.A., Hall J.A., Konczal J., Lilla S., McArthur D., McConnell P., McDonald L., McGarry L., McKinnon H., McMenemy C., Mezna M., Morrice N.A., Munro J., Naylor G., Rath N., Schüttelkopf A.W., Sime M, & Olson M.F. (2018). Discovery of potent and selective MRCK inhibitors with therapeutic effect on skin cancer. Cancer research, 78(8), 2096-2114.
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