To evaluate the serum antibody levels, AlphaLISA was used. Initially, AlphaLISA was performed in 384-well microtiter plates (white opaque OptiPlate, PerkinElmer, Waltham, MA, USA) containing either 2.5 µl of 1:100 diluted serum or 2.5 µl of 10 µg/ml of GST and GST-AP3D1 protein (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/ml dextran-500 and 0.05% Proclin-300). The reaction mixture was then incubated at room temperature for 6–8 h. Secondly, after adding anti-human IgG-conjugated acceptor beads (2.5 µl at 40 µg/ml) and glutathione-conjugated donor beads (2.5 µl at 40 µg/ml), the mixture was further incubated at room temperature in the dark for 7–14 days. Chemical emissions were read on an EnSpire Alpha microplate reader (PerkinElmer) as previously described18 (link)–23 (link),30 (link). Specific reactions were then calculated by subtracting the alpha photon counts of the GST and buffer control from the counts of the GST-AP3D1 protein.
Enspire alpha
Manufactured by PerkinElmer
Sourced in United States
The EnSpire Alpha is a multi-mode microplate reader designed for versatile applications in life science research. It offers a compact and configurable platform for various detection modes, including absorbance, fluorescence, and luminescence. The EnSpire Alpha provides researchers with a reliable and flexible solution for a wide range of analytical tasks in the laboratory.
Automatically generated - may contain errors
Lab products found in correlation
Optiplate, by PerkinElmer (8 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Britelite plus, by PerkinElmer (2 mentions)
E2620, by Promega (1 mentions)
Celltiter glo 2.0 luminescent cell viability assay, by Promega (1 mentions)
D300e digital dispenser, by Tecan (1 mentions)
α msh, by Merck Group (1 mentions)
Rpmi 1640, by US Biological (1 mentions)
Ultrapure dnase, by Thermo Fisher Scientific (1 mentions)
Black 96 well plate, by PerkinElmer (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Optiplate microtiter plates, by PerkinElmer (1 mentions)
Ldh release, by Promega (1 mentions)
Glucose assay kit, by Merck Group (1 mentions)
Aspergillus niger amyloglucosidase, by Merck Group (1 mentions)
Anti human cd3, by BD (1 mentions)
Arachidonic acid, by Merck Group (1 mentions)
Docosahexaenoic acid, by Merck Group (1 mentions)
Deferoxamine, by Cayman Chemical (1 mentions)
Stearic acid, by Cayman Chemical (1 mentions)
32 protocols using enspire alpha
1
Serum Antibody Quantification by AlphaLISA
2
Luciferase-based Cell Viability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A volume of 100 µl of luciferase expressing cells were transferred into 96-well plates. Cells were mixed with an equal volume of bright-glo assay solution (Promega, E2620) and incubated at room temperature for 10 min with shaking. Half of the mixture was transferred into a 96-well white opaque plate. The luminescence signal was read using an EnSpire alpha (PerkinElmer) detector.
3
Quantitative Serum Antibody Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the quantitative measurement of serum antibodies against the purified proteins, AlphaLISA was performed using 384-well microtiter plates (white opaque OptiPlate, Perkin Elmer) containing 2.5 μL of 1:100-diluted sera and 2.5 μL of GST or GST fusion proteins (10 μg/mL) in AlphaLISA buffer (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL Dextran-500, and 0.05% Proclin-300). The reaction mixture was incubated for 6-8 h at room temperature. Subsequently, anti-human IgG-conjugated acceptor beads (2.5 μL, 40 μg/mL) and glutathione-conjugated donor beads (2.5 μL, 40 μg/mL) were added, and the mixture incubated for 7-14 days at room temperature in the dark. The chemical emission was read on an EnSpire Alpha microplate reader (Perkin Elmer), as previously described [17 (link), 39 (link), 40 (link)-45 (link)]. Specific reactions were calculated by subtracting the Alpha values (Alpha counts) of the GST control from those of GST fusion proteins.
4
Adavosertib Dose-Response in Neuroblastoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Untreated SHEP-21N cells (750 cells/well) and SHEP-21N cells under doxycycline pressure for ≥3 days (1,000 cells/well) were seeded in 384-well microplates (Greiner Bio-One, #781080) and incubated overnight. Next, cells were exposed to 14.6 nmol/L to 15 μmol/L adavosertib in 2-fold serial dilutions using the Tecan D300e Digital Dispenser. DMSO and thonzonium bromide (20 μmol/L) were included as negative and positive control, respectively. Readout was conducted on days 1 and 3 after treatment initiation using the CellTiter-Glo 2.0 Luminescent Cell Viability Assay (Promega) and the EnSpire Alpha multimode plate reader (PerkinElmer) for luminescence detection. Cell viabilities were calculated as described for in vitro HTS and dose–response curves were generated using GraphPad Prism (version 9.3.1). All experiments were conducted in triplicate in three independent experiments. Each data point represents the mean ± standard deviation (SD). The DepMap GDSC dataset (https://depmap.org/portal/ ; ref. 30 (link)) was utilized to explore the efficacy of adavosertib (i.e., MK-1775) in an independent cohort. A one-sided Wilcoxon test was performed to statistically compare the AUC (31 (link)) difference between MYCN amplified (N = 6) versus MYCN wild-type (N = 14) classical neuroblastoma cell lines.
5
Investigating Hair Pigmentation Modulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the effect of pigmentation drugs on the hair color of generated shafts, the DMEM/F-12 medium was supplemented with 10 nM α-MSH (Merck Millipore, Burlington, MA, USA) on day 2 after seeding. Then, one-half of the DMEM/F-12 medium containing 10 nM α-MSH was changed every 2 days. The hair shaft color was observed using an all-in-one microscope and digital camera, and the relative expression levels of genes associated with hair pigmentation were evaluated via real-time reverse transcription PCR (RT-PCR) analysis. The total amount of generated melanin was quantified using a plate reader (EnSpire Alpha, PerkinElmer, Waltham, MA, USA).
6
Z-RVP-384 Assay Performance Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We developed several acceptance criteria based on Z-RVP-384 assay performance characterization: required assay signal (RLU) and positive and negative control ranges. Maximum signal (RLU) of the Z-RVP-384 assay must be greater than 100,000 RLU and less than 10,000,000 RLU, the detection limit of the EnSpire Alpha chemiluminescence readers we used (Perkin Elmer technical guidance). Acceptance ranges for the positive and negative serum controls included on each plate were established as mean log10EC50 ± 3x SD for the positive control and mean log10EC50 + 2x SD for the negative control. All samples on a failed plate must be retested.
7
Antifungal Susceptibility Testing of Candida Species
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Minimum inhibitory concentrations
(MICs) to the two Candida species were
determined following the guidelines of the European Committee on Antimicrobial
Susceptibility Testing (EUCAST).41 C. auris AR390 and C. albicans AR761 strains were grown in Yeast Peptone Dextrose overnight at
30 °C with shaking. The inoculum was prepared according to EUCAST
methods with modifications as follows: RPMI 1640 (US Biological R8998–07)
was supplemented with 2% glucose and buffered with morpholinepropanesulfonic
acid adjusted to pH 7.0. Hectoramide B (1 ) was serially
diluted in a 96-well plate and added at a final starting concentration
of 128 μg/mL. After 24 h, the absorbance was read at 530 nm
using an Enspire Alpha plate reader (PerkinElmer).
MIC to the S. cerevisiae ABC16-Monster strain were determined
using microtiter broth dilution in Yeast Peptone Dextrose (YPD) media.
Frozen spore suspensions of S. cerevisiae were grown on an overnight plate culture in YPD agar. Wells were
inoculated to a final concentration of 1.5 × 105 cfu/mL.
Well plates were incubated at 30 °C for 20 h, and the MICs were
defined as the lowest concentration of drug completely inhibiting
visible growth. Cycloheximide and fluconazole were used as positive
controls.
(MICs) to the two Candida species were
determined following the guidelines of the European Committee on Antimicrobial
Susceptibility Testing (EUCAST).41 C. auris AR390 and C. albicans AR761 strains were grown in Yeast Peptone Dextrose overnight at
30 °C with shaking. The inoculum was prepared according to EUCAST
methods with modifications as follows: RPMI 1640 (US Biological R8998–07)
was supplemented with 2% glucose and buffered with morpholinepropanesulfonic
acid adjusted to pH 7.0. Hectoramide B (
diluted in a 96-well plate and added at a final starting concentration
of 128 μg/mL. After 24 h, the absorbance was read at 530 nm
using an Enspire Alpha plate reader (PerkinElmer).
MIC to the S. cerevisiae ABC16-Monster strain were determined
using microtiter broth dilution in Yeast Peptone Dextrose (YPD) media.
Frozen spore suspensions of S. cerevisiae were grown on an overnight plate culture in YPD agar. Wells were
inoculated to a final concentration of 1.5 × 105 cfu/mL.
Well plates were incubated at 30 °C for 20 h, and the MICs were
defined as the lowest concentration of drug completely inhibiting
visible growth. Cycloheximide and fluconazole were used as positive
controls.
8
Measuring cGMP Levels in Stimulated Hippocampal Neurons
9
Quantitative Serum Antibody Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The serum antibodies against the purified proteins were quantitatively measured by AlphaLISA. After being prepared according to Perkin Elmer’s instructions (Waltham, MA) and our previous reports [27 (link), 30 , 33 (link), 34 (link)], the samples mixture was incubated for 14 days at room temperature in the dark. The chemical emission was read using the EnSpire Alpha microplate reader (Perkin Elmer). Specific reactions were calculated by subtracting the alpha values (alpha-photon counts) of the GST control from those of GST-fusion proteins.
10
Measuring Glucosidase Activity in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Snap-frozen tissues were homogenized in UltraPure DNase- and RNase-free distilled water (Thermo Fisher Scientific). Tissues were weighed, homogenized, and centrifuged for 20 min at 10,000 × g to collect the supernatant. The enzymatic reaction was set up using 10 μL of sample (plasma or tissue homogenate) diluted appropriately and 20 μL of substrate, 4-methylumbelliferone (4MU)α-D-glucoside, in black 96-well plates (PerkinElmer). The reaction mixture was incubated at 37°C for 1 hr and then stopped by adding 150 μL of sodium carbonate buffer (pH 10.5). A standard curve (0–2,500 pmol/μL of 4MU) was used to measure released fluorescent 4MU from the individual reaction mixture using the EnSpire Alpha plate reader (PerkinElmer) at 449 nm (emission) and 360 nm (excitation). The protein concentration of the clarified supernatant was quantified by BCA (Thermo Fisher Scientific). To calculate the GAA activity in tissues, the released 4MU concentration was divided by the sample protein concentration, and activity was reported as nanomoles per hour per milligram protein.
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!