Briefly, 20 μl reaction mixtures containing 10 nM FTSC-labeled RNA and the indicated amounts of protein in binding buffer (10 mM Tris–HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2, 2 mM dithiothreitol, 2.5 mM NaH2PO4, 15 mM NaCl, 4% glycerol) were incubated in 96-well Black PS HE microplate (Molecular Devices) for half an hour. Fluorescence polarization (FP) assay were then carried out in quadruplicate on a Synergy 4 multi-mode microplate reader (BioTek) to measure the polarization change. The equilibrium dissociation constant (Kd) was obtained by fitting the binding curves using KaleidaGraph (28 (link)).
Synergy 4 multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy 4 Multi-Mode Microplate Reader is a compact, high-performance microplate reader designed for a wide range of assay applications. It features a monochromator-based optical system that can measure absorbance, fluorescence, and luminescence in a 6- to 384-well microplate format.
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Lab products found in correlation
Mtase glo, by Promega (2 mentions)
Bioluminescence assay mtase glo, by Promega (2 mentions)
Alamar blue, by Thermo Fisher Scientific (2 mentions)
Calibur flow cytometer, by BD (2 mentions)
Inova 500 spectrometer, by Agilent Technologies (2 mentions)
Zetasizer nano zs90, by Malvern Panalytical (2 mentions)
Gen5ch 2, by Agilent Technologies (2 mentions)
Dowex 50wx4, by Merck Group (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Black walled 96 well plate, by Corning (1 mentions)
Actin polymerization kit, by Cytoskeleton (1 mentions)
Gen5 reader control and data analysis software, by Agilent Technologies (1 mentions)
Costar 96 well plate, by Corning (1 mentions)
Ova grade 5, by Merck Group (1 mentions)
Gpc system, by Malvern Panalytical (1 mentions)
Amplex red h2o2 assay kit, by Thermo Fisher Scientific (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Quant it ribogreen rna assay, by Thermo Fisher Scientific (1 mentions)
Alamarblue cell viability assay, by Thermo Fisher Scientific (1 mentions)
38 protocols using synergy 4 multi mode microplate reader
1
Fluorescence Polarization Binding Assay
2
Kinetic Analysis of SETD3 Methyltransferase
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Human beta-actin peptide (residues 66-80; TLKYPIEHGIVTNWD) was used as a
substrate for SETD3. The reaction mixture containing 20 mM Tris (pH 8.0), 50 mM
NaCl, 0.1 mg/ml BSA, 1 mM DTT, and 0.18 μM SETD3. To determine the Km
value for peptide, the concentration of S-adenosyl-L-methionine (SAM) was kept
constant at 40 μM, while for determination of the Km for SAM, peptide
concentration was kept constant at 50 μM. Reactions were carried out at
room temperature for 20 min with a total volume of 20 μl, and terminated
by the addition of trifluoroacetic acid (TFA) to 0.1% (v/v). The activity of
SETD3 was measured using a bioluminescence assay (MTase-Glo™, Promega) in
which the reaction by-product S-adenosyl-homocysteine (SAH) is converted into
ATP in a two-step reaction and can be detected via a luciferase
reaction37 (link). In
general, 5 μl of reaction mixture was transferred to a low-volume
384-well plate and the luminescence assay was performed according to the
manufacture’s protocol. A Synergy 4 Multi-Mode Microplate Reader (BioTek)
was used to measure luminescent signal.
substrate for SETD3. The reaction mixture containing 20 mM Tris (pH 8.0), 50 mM
NaCl, 0.1 mg/ml BSA, 1 mM DTT, and 0.18 μM SETD3. To determine the Km
value for peptide, the concentration of S-adenosyl-L-methionine (SAM) was kept
constant at 40 μM, while for determination of the Km for SAM, peptide
concentration was kept constant at 50 μM. Reactions were carried out at
room temperature for 20 min with a total volume of 20 μl, and terminated
by the addition of trifluoroacetic acid (TFA) to 0.1% (v/v). The activity of
SETD3 was measured using a bioluminescence assay (MTase-Glo™, Promega) in
which the reaction by-product S-adenosyl-homocysteine (SAH) is converted into
ATP in a two-step reaction and can be detected via a luciferase
reaction37 (link). In
general, 5 μl of reaction mixture was transferred to a low-volume
384-well plate and the luminescence assay was performed according to the
manufacture’s protocol. A Synergy 4 Multi-Mode Microplate Reader (BioTek)
was used to measure luminescent signal.
3
PEGylated Thermosensitive Nano-Liposomes for Doxorubicin Delivery
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PLDTS (PEGylated thermosensitive nano-liposomes remote-loaded with doxorubicin) were prepared according to previously reported methods [24] (link), [25] (link), [46] (link). Briefly, PLDTS were fabricated by lipid film hydration, followed by down-sizing using extrusion. Doxorubicin was remote loaded by transmembrane pH gradient using sodium citrate buffer as intra-liposome low-pH medium. Unloaded drug was removed by the cation exchange resin Dowex 50WX-4 (Sigma-Aldrich, St. Louis, MO, USA) or cation exchange column Stata-X-C Polymeric Strong Cation 200 mg/3 ml (Phenomenex, Torrence, CA, USA) [47] (link). The final amounts of intraliposomal doxorubicin were determined by using intensity of absorption at 480 nm using Synergy 4 Multi-Mode Microplate Reader (Biotek Instruments, Winooski, VT, USA), as previously described by Amselem et al. [48] (link).
4
CamA Methylation Assay Protocol
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The CamA
methylation assay was performed in 100 mM NaCl, 50 mM Tris-HCl, pH
7.5, 1 mM dithiothreitol (DTT), 0.25% DMSO, and 0.1 mg mL–1 BSA. CamA (50 nM final concentration) was preincubated with varied
concentration of compound at RT (∼22 °C) for 5 min followed
by addition of 2.5 μM double-stranded DNA (final concentration)
(Table S2 ) and 40 μM SAM (final concentration)
(Figures 1 B,D and S1 ). To determine the Ki of compound 11a against CamA, the methylation
reaction was carried out with varying concentrations of SAM (20, 30,
40, and 80 μM) and inhibitor11a (0, 0.25, and
0.5 μM) (Figure 2 E). Reactions were incubated for 3 min at ∼22 °C (RT),
and quenched by adding TFA to 0.1%. The final reaction mixture (5
μL) was transferred to a low-volume 384-well plate, and the
SAH concentration was measured by Promega luminescence assay (MTase-Glo).70 (link) A Synergy 4 Multi-Mode Microplate Reader (BioTek)
was used to detect the luminescence signal.
methylation assay was performed in 100 mM NaCl, 50 mM Tris-HCl, pH
7.5, 1 mM dithiothreitol (DTT), 0.25% DMSO, and 0.1 mg mL–1 BSA. CamA (50 nM final concentration) was preincubated with varied
concentration of compound at RT (∼22 °C) for 5 min followed
by addition of 2.5 μM double-stranded DNA (final concentration)
(
(
reaction was carried out with varying concentrations of SAM (20, 30,
40, and 80 μM) and inhibitor
0.5 μM) (
and quenched by adding TFA to 0.1%. The final reaction mixture (5
μL) was transferred to a low-volume 384-well plate, and the
SAH concentration was measured by Promega luminescence assay (MTase-Glo).70 (link) A Synergy 4 Multi-Mode Microplate Reader (BioTek)
was used to detect the luminescence signal.
5
DNA Methylation Activity Quantification
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The DNA methylation activity was measured using a Promega bioluminescence assay (MTase-Glo) (130 (link)) in which the generated SAH is converted to ATP in two steps, and the generated ATP is detected through a luciferase reaction. The concentration of SAH was calibrated based on the SAH standard in a linear regression plot of the luminescence signal of the Promega assay (see Fig. S3 of (131 (link)) or Fig. S1 of (48 (link))).
Typically, the methylation reaction was conducted at room temperature in a total volume of 20 μl in the optimal reaction buffer (100 mM NaCl, 20 mM Tris–HCl pH 7.5, 1 mM DTT). In general, 2 × (N4CMT and SAM) was preincubated at room temperature (∼22 °C) for about 3 min and the reaction was started by adding the same volume of 2 × DNA substrate. Reactions were stopped by adding TFA to a final concentration of 0.1% v/v. A 5 μl aliquot of the terminated reaction mixture was transferred to a 384 low-volume microwell plate, and the luminescence signal was measured with a Synergy 4 multimode microplate reader (BioTek).
Typically, the methylation reaction was conducted at room temperature in a total volume of 20 μl in the optimal reaction buffer (100 mM NaCl, 20 mM Tris–HCl pH 7.5, 1 mM DTT). In general, 2 × (N4CMT and SAM) was preincubated at room temperature (∼22 °C) for about 3 min and the reaction was started by adding the same volume of 2 × DNA substrate. Reactions were stopped by adding TFA to a final concentration of 0.1% v/v. A 5 μl aliquot of the terminated reaction mixture was transferred to a 384 low-volume microwell plate, and the luminescence signal was measured with a Synergy 4 multimode microplate reader (BioTek).
6
Cytotoxicity Evaluation of Peptides
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Material acute cytotoxicity was tested using human CPCs. The cells were stained with calcein-AM cell viability dye (25 nm; eBioscience) for 30 min and 500,000 cells were resuspended in 50 µL of each peptide (10 mM). After 15 min, 1 mL of growth media was added to the peptide-cell mixture and 50 µL was transferred into a 24-well plate for imaging. Five to six images were taken per condition using a fluorescence microscope (Carl Zeiss, Dublin, CA, USA). Cells resuspended in media only were used as a control. The percentage of viable cells was determined by counting the stained cells over the total number of cells. Three experiments were performed (n = 8–12 repeats in duplicate). Alternatively, CPCs were also cultured up to 4 days with or without peptides, and cytotoxicity was evaluated by an Alamar blue metabolic activity assay. In all, 3500 hCPCs were plated into each well of a 96-well plate in growth media. At 24 h after cell seeding, baseline metabolic activity was taken by incubating the cells in Alamar blue (1:10 in medium, Invitrogen) for 3 h and analyzing at 550/585 nm with a Synergy™ 4 Multi-Mode Microplate Reader (Biotek). Peptide was added to cells and cultured for 4 days. Alamar blue assay was repeated after 24 and 96 h, and growth media was used as control (n = 3 repeats in quadruplicate). Metabolic activity reported as fold increase vs baseline values.
7
Methylation Assays of PCIF1, MettL3-MettL14, and HemK2-Trm112
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Methylation assays of PCIF1 on different
RNA and DNA oligonucleotides were conducted in the reaction buffer
containing 20 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, 20
μM, or 100 μM S-adenosyl-l -methionine
(SAM), with various PCIF1 and substrate concentrations, at 37 °C
(for reaction time up to 4 h) or at room temperature (∼22 °C
overnight). Methylation assays of MettL3-MettL14 (NCBI: NP_062826.2
and NP_066012.1) and HemK2-Trm112 (NCBI: NP_037372.4 and NP_057488.1)
were conducted under the conditions as previously characterized in
our laboratory.18 (link),25 (link),26 (link),50 (link)To calibrate SAH concentration and
luminescence, the SAH standard solution within the MTase-Glo Methylation
Assay Kit (Promega) was subjected to serial twofold dilutions, starting
from 4 μM. Luminescence signals from a standard concentration
curve of SAH were generated according to the manufacturer’s
protocol. A 5 μL aliquot of sample was transferred into a low-volume
384-well plate, and the luminescence signal was detected using a Synergy
4 Multi-Mode microplate reader (BioTek). A linear regression of the
SAH standard was plotted against luminescence.
RNA and DNA oligonucleotides were conducted in the reaction buffer
containing 20 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, 20
μM, or 100 μM S-adenosyl-
(SAM), with various PCIF1 and substrate concentrations, at 37 °C
(for reaction time up to 4 h) or at room temperature (∼22 °C
overnight). Methylation assays of MettL3-MettL14 (NCBI: NP_062826.2
and NP_066012.1) and HemK2-Trm112 (NCBI: NP_037372.4 and NP_057488.1)
were conducted under the conditions as previously characterized in
our laboratory.18 (link),25 (link),26 (link),50 (link)To calibrate SAH concentration and
luminescence, the SAH standard solution within the MTase-Glo Methylation
Assay Kit (Promega) was subjected to serial twofold dilutions, starting
from 4 μM. Luminescence signals from a standard concentration
curve of SAH were generated according to the manufacturer’s
protocol. A 5 μL aliquot of sample was transferred into a low-volume
384-well plate, and the luminescence signal was detected using a Synergy
4 Multi-Mode microplate reader (BioTek). A linear regression of the
SAH standard was plotted against luminescence.
8
Characterization of Polymeric Nanoparticles
9
Intracellular Calcium Dynamics in RyR2-KO Cells
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INS-1 and RyR2KO cells were plated at 70–90% confluency in black-walled 96-well plates (Corning) in RPMI-1640 media and incubated overnight at 37 °C, 5% CO2. Cells were washed twice with PBS and incubated with 100 μL 5 μM Fura-2 AM in KRBH for 1 h at room temperature. The KRBH containing Fura-2 AM was removed, the cells were washed twice with KRBH, and equilibrated for 30 min in 100 μL KRBH at room temperature. Cells were stimulated by injection of 100 μL 10 mM caffeine (2x) or KRBH (buffer control). Changes in intracellular Ca2+ concentrations were measured by recording the ratio of fluorescence intensities at 508/20 nm resulting from excitation of Fura-2 AM at 340/11 nm or 380/20 nm (center/bandpass) using a Synergy 4 multimode microplate reader (BioTek). Ratios were acquired every 0.7 s for 15 s before injection and 2 min after injection. Data were corrected for injection artifact by subtracting the change in fluorescence ratio measured in cells injected with KRBH alone.
10
Actin Polymerization Assay Protocol
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Actin Polymerization Assays were performed using a modified assay with the actin polymerization kit from Cytoskeleton Inc. (Denver, CO). The modified assay used G-buffer by combining 10 mL of General Actin Buffer with 40 µL ATP. Actin buffer (AB) was prepared by adding 50 µL of 20 µg/µL actin with 2.25 mL of G-buffer. Actin oligomers were depolymerized by incubating AB on ice for 60 min and centrifuged at 20,000×g for 30 min at 4 °C. Reactions were setup in black 96-well plates (corning) using 65 µLG-buffer, 10 µL AB, and 25 µL of 1 µM αB-crystallin protein or PBS control. Assays were started by adding 12 µL of actin polymerization buffer (500 mM KCl, 20 mM MgCl2, 50 mM guanidine carbonate, and 10 mM ATP). Wells were monitored for 60 min at excitation (λ 350 nm) and emission (λ 407 nm) on a Synergy 4 Multi-Mode Microplate Reader and Gen5 Reader Control and Data Analysis Software (BioTek, Winooski, VT). Data were plotted and analyzed statistically by ANOVA on repeated measure with Tukey's multiple comparison with GraphPad Prism (La Jolla, CA).
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