The effect of UNC4976, UNC3866, and UNC4219 on cell viability was determined using CellTiter-Glo (Promega #7573). Compound stocks at 50 mM in DMSO were diluted to 500 μM (or 5×) in PBS, yielding a DMSO concentration of 1%. A three-fold dilution series of ten total points was then generated in PBS + 1% DMSO. 5 μL of each 5× compound stock was plated in assay wells on a Corning 384-well, white-walled, clear-bottom, cell culture treated assay plate in technical triplicate. Mouse embryonic stem cells (mESCs) were harvested, counted, and diluted to a density of 5,000 cells/20 μL. 20 μL of cell suspension per well was added on top of pre-plated 5× compound stocks to generate 1× concentrations (0.2% DMSO). The assay plate was centrifuged for 30 s and then incubated for 48 hr at 37°C and 5% CO2. Wells without cells (media only) were included as negative controls. Following incubation at 37°C, the assay plate was equilibrated at room temperature along with the CellTiter-Glo reagent. 25 μL of CellTiter-Glo reagent was added to appropriate wells and the plate was centrifuged for 30 s. The assay plate was then placed on a plate shaker at room temperature for 2 min, and then allowed to equilibrate on a bench top for an additional 10 min. Luminescence was read on a PerkinElmer EnSpire Alpha Multimode Plate Reader.
Enspire alpha multimode plate reader
Manufactured by PerkinElmer
Sourced in United States
The EnSpire Alpha Multimode Plate Reader is a versatile laboratory instrument designed for a wide range of detection technologies. It can perform absorbance, fluorescence, and luminescence measurements on microplates.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo, by Promega (2 mentions)
Alphaplate, by PerkinElmer (1 mentions)
Recombinant human lifr his tag, by Sino Biological (1 mentions)
Biotinylated recombinant human lif, by Sino Biological (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Polymorphprep, by Axis-Shield (1 mentions)
Steady glo luciferase assay system reagent, by Promega (1 mentions)
11 protocols using enspire alpha multimode plate reader
1
Cell Viability Assay for mESC Compounds
2
ROS Generation Quantification in Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils (2 × 106 cells/ml) were incubated for 20 min at 37°C with gentle agitation in Ca2+/Mg2+-free Hanks’ Balanced Salt Solution (HBSS) with 10 μM 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA; Thermo Fisher Scientific). Following a 5-min centrifugation at 1200 rpm, cells were resuspended in HBSS with Ca2+/Mg2+ at a density of 2 × 106 cells/ml with or without propofol or lipid emulsion. After a 30-min incubation at 37°C, cells were seeded in 96-well plates at a density of 2 × 105 cells/well. PMA was added to applicable wells, and ROS generation quantified using an EnSpire Alpha Multimode Plate Reader (PerkinElmer), with reads collected every 15 min for 2 h (485 nm excitation, 530 nm emission). Between reads, plates were incubated and protected from light at 37°C with 5% CO2. To account for variability between donors and dye loading between experiments, data were expressed as percentages of the maximum fluorescence intensity detected within each experimental repeat.
3
Neutrophil Extracellular Trap Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils were incubated for 30 min with propofol at a concentration of 5 μg/ml or a corresponding concentration of lipid emulsion (adjusted to soybean oil content). Cells were then treated with either 25 nM PMA for 4 h or nigericin for 3 h at 37°C with 5% CO2, followed by addition of micrococcal nuclease (1 U/mL; Dako Denmark). After an additional 10-min incubation at 37°C, enzymatic activity was stopped via addition of 10 mM EDTA. Cells were then centrifuged for 5 min at 200 ×g, and extracellular DNA in supernatant samples quantified using the Quant-iT PICO GreenTM dsDNA Assay Kit (Invitrogen) and an EnSpire Alpha Multimode Plate Reader (PerkinElmer).
4
LIFR/LIF Binding Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human LIFR (His Tag) and biotinylated recombinant human LIF were purchased from Sino Biologicals (Sino Biological Europe GmbH, Dusseldorf, Germany) and R&D Systems (Abingdon, UK), respectively, and both were reconstituted as required by the manufacturer.
Inhibition of LIFR/LIF binding by mifepristone was measured by Alpha Screen (Amplified Luminescent Proximity Homogeneous Assay). The assay was performed in white, low-volume, 384-well AlphaPlates (PerkinElmer, Waltham, MA, USA) using a final volume of 25 μL and an assay buffer containing 25 mM Hepes (pH 7.4), 100 mM NaCl, and 0.005% Kathon. The concentration of DMSO in each well was maintained at 5% vol/vol.
LIFR (His Tag, final concentration 4.5 nM) was incubated with either mifepristone (10 concentrations, from 4.12 nM to 200 µM) or a vehicle for 45 min under continuous shaking. Then, LIF was added (biotinylated, final concentration 20 nM), and the samples were incubated for 15 min prior to adding His-Tag acceptor beads (final concentration 20 ng/µL) for 30 min. Then, streptavidin donor beads were added (final concentration 20 ng/µL), and the plate was incubated in the dark for 3 h and then read in an EnSpire Alpha multimode plate reader (PerkinElmer, Waltham, MA, USA).
Inhibition of LIFR/LIF binding by mifepristone was measured by Alpha Screen (Amplified Luminescent Proximity Homogeneous Assay). The assay was performed in white, low-volume, 384-well AlphaPlates (PerkinElmer, Waltham, MA, USA) using a final volume of 25 μL and an assay buffer containing 25 mM Hepes (pH 7.4), 100 mM NaCl, and 0.005% Kathon. The concentration of DMSO in each well was maintained at 5% vol/vol.
LIFR (His Tag, final concentration 4.5 nM) was incubated with either mifepristone (10 concentrations, from 4.12 nM to 200 µM) or a vehicle for 45 min under continuous shaking. Then, LIF was added (biotinylated, final concentration 20 nM), and the samples were incubated for 15 min prior to adding His-Tag acceptor beads (final concentration 20 ng/µL) for 30 min. Then, streptavidin donor beads were added (final concentration 20 ng/µL), and the plate was incubated in the dark for 3 h and then read in an EnSpire Alpha multimode plate reader (PerkinElmer, Waltham, MA, USA).
5
Antibiotic Synergy Evaluation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacteria were cultured in the same media throughout (CA-MHB or RPMI+) prior to the addition of antibiotics. Mid-logarithmic phase cultures were diluted to approximately 5 × 105 CFU (~OD600 = 0.002) and 180 μl added to each experimental well of a 96 well flat bottom plate. Either 20 μl of 1xDPBS or 20 μl of the desired 10× drug stock were added to each well. Plates were incubated shaking at 100 rpm at 37 °C overnight. Bacterial growth (OD600) was determined approximately 20 h later with an Enspire Alpha multimode plate reader (PerkinElmer). To calculate the MIC90, defined as the amount of drug required to inhibit ≥90% of the growth of the untreated controls, the density of each drug-treated well was compared to untreated control. Determination of synergy was performed in the same manner except combinations of AZM and MIN were examined using checkerboard assays, where 10 μl from 20× stocks of each drug were added to each well so that the ratio of media to drugs remained consistent to the monotherapy MIC experiments. Fractional inhibition concentrations (FIC) were determined for each drug in combination and a fractional inhibition concentration index (FICI) value was calculated. Synergy was defined as an FICI value ≤0.5 [18 (link)].
6
SETD2 and Set2 Histone Methyltransferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For an 8-µL HMT reaction, 0.2 µM wild-type or mutant SETD2 and Set2 proteins, 20 µM SAM, and 2 µM NCP were mixed in the buffer of 50 mM Tris–HCl, pH 8.0, 2 mM DTT, 5% glycerol, and 0.4 mg/mL BSA, and were incubated at 30 °C for 2 and 3 h, respectively. The reaction was stopped by adding 2 μL of 0.5% trifluoroacetic acid (TFA) and the HMT activity was evaluated using an MTase-GloTM Methyltransferases Assay Kit (Promega). The luminescent signal that corresponds to the production of SAH was measured using the EnSpire Alpha Multimode plate reader (PerkinElmer) in a white 384-well plate. Each reaction was run in triplicate and was reported as the means ± standard deviation (SD).
For evaluation of the activity specificity of SETD2 and Set2, the HMT reaction mixtures were separated on a 15% SDS–PAGE gel, and the mono-, di- and tri-methylation of histone H3K36 was detected by western blotting using the corresponding antibodies (H3K36me1 antibody: #A2364, ABclonal; H3K36me2 antibody: #A2365, ABclonal; H3K36me3 antibody: #A2366, ABclonal).
For evaluation of the activity specificity of SETD2 and Set2, the HMT reaction mixtures were separated on a 15% SDS–PAGE gel, and the mono-, di- and tri-methylation of histone H3K36 was detected by western blotting using the corresponding antibodies (H3K36me1 antibody: #A2364, ABclonal; H3K36me2 antibody: #A2365, ABclonal; H3K36me3 antibody: #A2366, ABclonal).
7
Measuring Transcriptional Activity with Luciferase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The luciferase activity of transfected cells was measured using the Luciferase/β-Galactosidase Luciferase Assay Kit, Dual-Light (Applied Biosystems). The relative transcriptional activity was calculated as Firefly Luc/β-galactosidase. The analysis was performed in quadruplicate, and the results are reported as the ratio of firefly luciferase activity and β-galactosidase (relative transcriptional activity). The light emission was measured by EnSpire Alpha Multimode Plate Reader (PerkinElmer).
8
Cell Viability Assay with UNC Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effect of UNC6261 and UNC7394 on cell viability was determined using CellTiter‐Glo (Promega #7573). Compound stocks at 50 mm in DMSO were diluted to 500 µm (or 5×) in PBS, yielding a DMSO concentration of 1%. A threefold dilution series of ten total points was then generated in PBS + 1% DMSO. 5 µL of each 5× compound stock was plated in assay wells on a Corning 384‐well, white‐walled, clear‐bottom, cell culture treated assay plate in technical triplicate. U2OS Cells were harvested, counted, and diluted to a density of 5000 cells per 20 µL. 20 µL of cell suspension per well was added on top of preplated 5× compound stocks to generate 1× concentrations (0.2% DMSO). The assay plate was centrifuged for 30 s and then incubated for 48 h at 37 °C and 5% CO2. Wells without cells (media only) were included as negative controls. Following incubation at 37 °C, the assay plate was equilibrated at room temperature along with the CellTiter‐Glo reagent. 25 µL of CellTiter‐Glo reagent was added to appropriate wells and the plate was centrifuged for 30 s. The assay plate was then placed on a plate shaker at room temperature for 2 min, and then allowed to equilibrate on a bench top for an additional 10 min. Luminescence was read on a PerkinElmer EnSpire Alpha Multimode Plate Reader.
9
Quantification of Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Under approval from UC San Diego IRB/HRPP and BCM IRB, venous blood was obtained after informed consent from healthy adult volunteers, with heparin as an anticoagulant. Neutrophils were isolated using PolymorphPrepTM (Axis-Shield) to create a density gradient by centrifugation according to the manufacturer's instructions. Fluorescence-based quantification of neutrophil extracellular traps (NETs) was performed as described previously(20 (link)). Briefly, isolated neutrophils were plated on 96-well tissue culture plates at 2 × 105 cells/well. Cells were pretreated with 50 μg/mL of purified human THP or mock-treated, and incubated at 37°C in 5% CO2 for 30 min, and then incubated for an additional 3 h with phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich) PMA (25 nM) to induce NET production(20 (link)). Micrococcal nuclease was then added at a final concentration of 500 mU/mL for 10 min to digest extracellular DNA. Plates were centrifuged at 200 g for 8 min; sample supernatant was then collected and transferred to a new 96-well plate. DNA was quantified using a Quant-iT PicoGreen® dsDNA Assay Kit from Life Technologies (Carlsbad, CA, USA), with fluorescence detected on intensity (485 nm excitation and 530 nm emission) measured by an EnSpire Alpha Multimode Plate Reader (PerkinElmer).
10
Luminescence-Based GBS Infection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
VK2-HRE and RAW-HRE cells were plated the day before in white opaque, tissue culture 96 well plates in culture media as described above. Cells were pretreated with 12 μg/mL AKB-4924 or vehicle for 4 h before infection. Cells were infected with GBS COH1 or isogenic ΔcylE at MOI = 20 as indicated in figure legends. At 2 h postinfection, cells were washed once with PBS and lysed with 25 μL/well of Steady-Glo® Luciferase Assay System reagent (Promega) for 5 min at room temp. RPMI-1640, containing no phenol red, was added (25 μL/well), mixed, incubated at room temperature for 15 min, and luminescence was then measured on an EnSpire Alpha Multimode Plate Reader (PerkinElmer). Arbitrary units of luminescence were normalized across individual experiments using values from vehicle-treated, uninfected VK2-HRE or RAW-HRE cells.
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!