Cell proliferation was assessed using the Delfia Cell Proliferation kit (Caliper PerkinElmer Life Sciences, Waltham, MA). Approximately 1 × 104 cells were seeded into a 96-well Isoplate (PerkinElmer Life Sciences) and grown in complete media. Bromodeoxyuridine (BrdUrd, 10 μM ) was added to the medium 24 hr prior to the end of the assay. The cells were subsequently fixed, the DNA was denatured, and the amount of incorporated BrdUrd was determined using an Eu-labeled monoclonal anti-BrdU antibody. Following formation of highly fluorescent chelates, fluorescent signal was detected by time-resolved fluorometry using a Victor3 (link) multilabel reader (PerkinElmer Life Sciences). The same number of cells was seeded on a separate 96-well Isoplate and grown overnight before fixation with 4% paraformaldehyde and nuclear staining with DAPI. DAPI fluorescence was detected also using a Victor3 (link) multilabel reader. Final results are reported as Eu fluorescence normalized to DAPI signal.
Isoplate
Manufactured by PerkinElmer
Sourced in Australia, United States, Germany, United Kingdom
The Isoplate is a laboratory instrument designed for high-throughput sample preparation and analysis. It provides a platform for efficient and consistent processing of multiple samples simultaneously. The Isoplate is a versatile tool that can be used for a variety of applications in research and diagnostic laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Infinite f500, by Tecan (3 mentions)
Microbeta2 plate reader, by PerkinElmer (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
3h glycine, by Merck Group (2 mentions)
3h glycine, by PerkinElmer (2 mentions)
3h diprenorphine, by PerkinElmer (1 mentions)
Microbeta counter, by PerkinElmer (1 mentions)
Microscint e, by PerkinElmer (1 mentions)
Bz 9000, by Keyence (1 mentions)
Hoechst 33342 staining, by Dojindo Laboratories (1 mentions)
Liperfluo reagent, by Dojindo Laboratories (1 mentions)
Hydrop, by Goryo Chemical (1 mentions)
Filtermax f5, by Molecular Devices (1 mentions)
Cary 50 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Microbeta trilux scintillation counter, by PerkinElmer (1 mentions)
35s gtpγs, by PerkinElmer (1 mentions)
Microscint scintillation liquid, by PerkinElmer (1 mentions)
Microbeta plate reader, by PerkinElmer (1 mentions)
Optiphase supermix, by PerkinElmer (1 mentions)
Poly d lysine hydrobromide, by Merck Group (1 mentions)
14 protocols using isoplate
1
Quantitative Cell Proliferation Assay
2
Inhibition of Amyloid-Beta Aggregation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aβ1-42 were dissolved in 10 mM NaOH, H2O, and PB 50 mM (1:1:2) to 2.5 μM in the absence or presence of the cinnamon extracts or the fractions (2.5 μg/ml) and were incubated at 37°C in 20 µM ThT (Sigma) in 96 well black plates (Isoplate, Perkin Elmer). A plate reader monitored the ThT fluorescence for 24 h by a plate reader (Infinite F500 Tecan: excitation 448 nm, emission 485 nm, 37°C). Data were expressed as the mean of three replicates, calculated by subtracting the relative control solutions (extracts or fractions alone), and expressed as the percentage of reduction of Aβ1-42 aggregation.
3
Radioligand Binding Assay for Opioid Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells
were seeded at
50 000 per well in a 96-well Isoplate (PerkinElmer, Melbourne,
Australia), allowed to adhere for 6 h, and then serum starved overnight.
Plates were washed once with ice-cold assay buffer (146 mM NaCl, 10
mMd -glucose, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 1.5 mM NaHCO3, 10 mM HEPES, pH 7.4). Cells were
incubated with increasing concentrations of SNC-80 (in the absence
or presence of increasing concentrations of allosteric modulator)
for 4 h at 4 °C in the presence of 0.3 nM [3H]-diprenorphine
(PerkinElmer, specific activity 36.1 Ci/mmol). Nonspecific binding
was determined by the coaddition of 100 μM naloxone. After washing
in cold saline, cells were solubilized in Optiphase scintillant, and
radioactivity was measured in a MicroBeta counter (PerkinElmer).
were seeded at
50 000 per well in a 96-well Isoplate (PerkinElmer, Melbourne,
Australia), allowed to adhere for 6 h, and then serum starved overnight.
Plates were washed once with ice-cold assay buffer (146 mM NaCl, 10
mM
incubated with increasing concentrations of SNC-80 (in the absence
or presence of increasing concentrations of allosteric modulator)
for 4 h at 4 °C in the presence of 0.3 nM [3H]-diprenorphine
(PerkinElmer, specific activity 36.1 Ci/mmol). Nonspecific binding
was determined by the coaddition of 100 μM naloxone. After washing
in cold saline, cells were solubilized in Optiphase scintillant, and
radioactivity was measured in a MicroBeta counter (PerkinElmer).
4
Lipogenesis and Phospho-ACC Assay in Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary rat hepatocytes were isolated and plated in 96 well plates at a density of 35,000 cells/well in Williams E Media for studies the following day. Cryopreserved human (Corning) or cyno monkey hepatocytes (Thermo Fisher Scientific) were plated at a density of 35,000 cells/well in Williams E Media for studies the following day. For phospho-ACC measurements cells were treated with compounds for 1 h and lysed in cell lysis buffer for subsequent MSD ELISA measurements (as described above). For de novo lipogenesis measurements, cells were treated with compounds for 1 h followed by treatment with compound and supplemented with 0.8 μCi 14C-2-acetic acid (ARC0158B, American Radiolabeled Chemicals) and incubated (37 °C, 5% CO2) for 1 h. After final incubation, experimental media was removed and 50 μL Cell Lytic MEM Protein Extraction (Sigma, CE0050) added to each well then placed in −80 °C freezer until processing. For processing, lysates were thawed on ice then 150 μL Microscint E (Perkin Elmer, 6013661) added to each condition than 200 μL transferred to a 96-well Isoplate (Perkin Elmer, 6005040) for counting.
5
Amyloid-β Aggregation Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aβ1-42
was dissolved in 10 mM NaOH, H2O, and 50 mM PB (1:1:2)
to 2.5 μM with or without a defined concentration of hop extracts
(0.25 mg/mL) or enriched fractions (0.03 mg/mL for fraction B and
0.0125 mg/mL for fractions B1, B2, B4, and D) and were incubated at
37 °C in 20 μM ThT (Sigma-Aldrich, St. Louis, MO) in 96-well
black plates (Isoplate, Perkin Elmer, Waltham, MA). The ThT fluorescence
was monitored for 24 h with a plate reader (Infinite F500 Tecan: excitation
448 nm, emission 485 nm, 37 °C). Data were expressed as the mean
of three replicates, calculated by subtracting the relative control
solutions (extract or fraction alone), and were expressed as the percentage
reduction of Aβ1-42 aggregation.
was dissolved in 10 mM NaOH, H2O, and 50 mM PB (1:1:2)
to 2.5 μM with or without a defined concentration of hop extracts
(0.25 mg/mL) or enriched fractions (0.03 mg/mL for fraction B and
0.0125 mg/mL for fractions B1, B2, B4, and D) and were incubated at
37 °C in 20 μM ThT (Sigma-Aldrich, St. Louis, MO) in 96-well
black plates (Isoplate, Perkin Elmer, Waltham, MA). The ThT fluorescence
was monitored for 24 h with a plate reader (Infinite F500 Tecan: excitation
448 nm, emission 485 nm, 37 °C). Data were expressed as the mean
of three replicates, calculated by subtracting the relative control
solutions (extract or fraction alone), and were expressed as the percentage
reduction of Aβ1-42 aggregation.
6
Fluorescent Probes for ROS Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Production of hydroxyl radical ( Á OH) and hypochlorous acid (HClO) were measured using the OxiORANGE reagent (Goryo Chemical, Sapporo, Japan). H 2 O 2 and NO were detected by HYDROP and diaminofluorescein-FM diacetate (DAF-FM DA) (Goryo Chemical), respectively. Cells plated in a 96-well Black IsoPlate (PerkinElmer, Waltham, MA, USA) were incubated with 0.5 µM OxiORANGE, 1 µM HYDROP, or 1 µM DAF-FM DA diluted in EMEM containing FBS for 30 min at 37 °C, and then washed with 100 µL Hank's balanced salt solution (HBSS) once. Fluorescence (Ex/Em = 535/595 nm for OxiORANGE, 485/ 535 nm for HYDROP and DAF-FM DA) was measured by using FilterMax F5 (Molecular Devices, San Jose, CA, USA). Normalized values were obtained by dividing the fluorescent intensities of HYDROP by protein concentration, those of DAF-FM DA by nuclear signal, and those of Oxi-ORANGE by nuclei number determined by Hoechst 33342 staining (Dojindo). Lipid peroxide was measured using the Liperfluo reagent (Dojindo). Cells were stained with 1 µM Liperfluo reagent diluted in EMEM without FBS for 30 min at 37 °C, and then observed under an all-in-one fluorescence microscope BZ-9000 (Keyence, Osaka, Japan).
7
Screening Coffee Extracts for Anti-Amyloid Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aβ1-42 were dissolved in 10 mM NaOH, H2O and PB 50 mM (1:1:2) to 2.5 M with or without coffee extracts (62.5 g/mL), 5-CQA (100 M, 35.4 μg/mL) or HMW and LMW fractions (62.5 g/mL), and were incubated at 37 °C in 20 µM ThT (Sigma, PB 50 mM, pH 7.4) in 96-well black plates (Isoplate, Perkin Elmer). The ThT fluorescence was monitored for 24 hours by a plate reader (Infinite F500 Tecan: excitation 448 nm, emission 485 nm, 37 °C). The corresponding curves are reported in Supplementary Material -Figure S1. Data were expressed as the mean of three replicates, calculated by subtracting the relative control solutions (extracts and compounds alone) and were expressed as the percentage reduction of Aβ1-42 aggregation. The UV-Vis absorption spectra (Varian Cary ® 50 UV-Vis spectrophotometer) of control solutions are reported in Supplementary Material -Figure S2.
8
G Protein Activation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SPAs were performed as previously described [45 (link)]. Briefly, cell membrane homogenates were incubated in 96-well Isoplates (Perkin Elmer Life Sciences, Maanstraat, Germany) in incubation buffer containing 0.4 nM [35S] GTPγS (Perkin Elmer) and 50 or 100 μM GDP for Gq/11 or G12/13 proteins, respectively. Specific antibodies for each Gα subunit (rabbit polyclonal anti-Gαq/11 and rabbit polyclonal anti-Gα12/13; Santa Cruz Biotechnologies, Madrid, Spain) and PVT SPA beads coated with protein A (Perkin Elmer) were used. Radioactivity was quantified on a MicroBeta TriLux scintillation counter (Perkin Elmer).
9
Saturation Binding Assay for mAChR Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FlpIn CHO cells
stably expressing the hemaglutinine (HA)-tagged wildtype human M1
or M4 (hM1 or M4 WT) mAChRs were plated at the density of 20 000,
and the human M1 DREADD (hM1Dq) or M4 DREADD (hM4Di) mAChRs were plated
at 50 000 cells per well of 96-well white clear-bottomed Isoplates
(PerkinElmer Life Sciences, Boston, MA), and grown overnight.
Saturation binding assays were performed to estimate the expression
levels (Bmax) and equilibrium dissociation
constant of the radioligand (Kd). Cells
were washed twice with phosphate-buffered saline (PBS), and incubated
with 0.03–10 nM [3H]NMS in a final volume of 100
μL of buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4) for 4 h at room temperature. Atropine at the final concentration
of 100 μM was used to determine nonspecific binding.
For
equilibrium binding assays, cells were incubated with increasing
concentrations of the ligands or 100 μM atropine, for nonspecific
binding, in the presence of Kd concentration
of the radioligand for 4 h at room temperature. The assays were terminated
by rapid removal of the radioligand, and two washes with 100 μL/well
ice-cold 0.9% NaCl buffer. Radioactivity was determined by addition
of 100 μL/well MicroScint scintillation liquid (PerkinElmer
Life Sciences, Boston, MA) and counted on a MicroBeta plate reader
(PerkinElmer Life Sciences, Boston, MA).
stably expressing the hemaglutinine (HA)-tagged wildtype human M1
or M4 (hM1 or M4 WT) mAChRs were plated at the density of 20 000,
and the human M1 DREADD (hM1Dq) or M4 DREADD (hM4Di) mAChRs were plated
at 50 000 cells per well of 96-well white clear-bottomed Isoplates
(PerkinElmer Life Sciences, Boston, MA), and grown overnight.
Saturation binding assays were performed to estimate the expression
levels (Bmax) and equilibrium dissociation
constant of the radioligand (Kd). Cells
were washed twice with phosphate-buffered saline (PBS), and incubated
with 0.03–10 nM [3H]NMS in a final volume of 100
μL of buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4) for 4 h at room temperature. Atropine at the final concentration
of 100 μM was used to determine nonspecific binding.
For
equilibrium binding assays, cells were incubated with increasing
concentrations of the ligands or 100 μM atropine, for nonspecific
binding, in the presence of Kd concentration
of the radioligand for 4 h at room temperature. The assays were terminated
by rapid removal of the radioligand, and two washes with 100 μL/well
ice-cold 0.9% NaCl buffer. Radioactivity was determined by addition
of 100 μL/well MicroScint scintillation liquid (PerkinElmer
Life Sciences, Boston, MA) and counted on a MicroBeta plate reader
(PerkinElmer Life Sciences, Boston, MA).
10
Glycine Release Assay in Spinal Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glycine release was assessed in spinal cord neurons at DIV 20–23. Spinal cord neurons were treated with a concentration range of BoNT/A (List Biological Laboratories, Campbell, CA, USA), BoNT/F1 (Metabiologics), or BoNT/FA (30 pM–300 aM) for 24 h at 37 °C. Following removal of neurotoxin, cells were briefly washed 3 times in HEPES-buffered salt solution (HBS) (136 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.2). Cells were loaded with 2 μCi/mL [3H]-glycine (Perkin Elmer) in HBS for 60 min at 35 °C. Following removal of [3H]-glycine, cells were briefly washed 3 times with HBS. Basal and stimulated [3H]-glycine release were established by incubation at 35 °C for 5 min with 50 μL/well assay media containing low potassium (3 mM KCl), or high potassium (60 mM KCl) HBS solutions, respectively. To determine retained [3H]-glycine, cells were lysed by adding 50 μL/well RIPA buffer (Sigma-Aldrich). Superfusates and cell lysates were transferred into 96-well Isoplates (Perkin Elmer) and 200 μL/well OptiPhase Supermix scintillation fluid was added. Radioactivity was quantified using a MicroBeta2 plate reader (Perkin Elmer).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!