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Furimazine

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Most cited protocols related to «Furimazine»

1
Coelenterazine-based Luminescence Screening
Cited 53 times
Random libraries were generated
by error-prone PCR (average of 2–3 mutations per clone). Library
1 (phase 1; template = Oluc-N166R) was screened (4,400 variants) with
coelenterazine. Library 2 (phase 2; template = C1A4E) was screened
(4,400 variants) with 11 novel coelenterazine analogues: 3840, 3841,
3842, 3857, 3880, 3881, 3886, 3887, 3889, 3897, and 3900 (Supplementary Figure s4). The 11 analogues represented
substitutions at positions 2, 6, and 8 and were considered to be representative
of the entire set of 24 compounds; 2,200 variants were screened with
compounds 3896 and 3894 (Supplementary Figure
s4). All hits (improved luminescence) were screened again with
the remaining coelenterazine analogues. Library 3 (phase 3; template
= C1A4E + Q18L/K33N/F54I/F68Y/L72Q/M75K/I90V) was screened in the
context of a mouse Id-X-HaloTag (where X = library) using coelenterazine
and furimazine (Figure 1c). Library screens
were performed on a Freedom robotic workstation (Tecan) as follows:
induced bacterial cultures (in 96-well microtiter plates) were lysed
with a buffer containing 300 mM HEPES pH 8, 200 mM thiourea, 0.3X
Passive Lysis Buffer (PLB, Promega), 0.3 mg mL–1 lysozyme, and 0.002 units of RQ1 DNase (Promega). Assay reagent
containing 1 mM CDTA, 150 mM KCl, 10 mM DTT, 0.5% (v/v) Tergitol,
and 20 μM substrate was then added to equal volumes of lysate.
Samples were measured on a GENios Pro luminometer (Tecan). Secondary
screening to confirm hits (defined as those variants producing greater
luminescence compared to that of the parental clone) and to test combination
sequences was completed using a similar protocol but in manual fashion
and in triplicate.
Hall M.P., Unch J., Binkowski B.F., Valley M.P., Butler B.L., Wood M.G., Otto P., Zimmerman K., Vidugiris G., Machleidt T., Robers M.B., Benink H.A., Eggers C.T., Slater M.R., Meisenheimer P.L., Klaubert D.H., Fan F., Encell L.P, & Wood K.V. (2012). Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate. ACS Chemical Biology, 7(11), 1848-1857.
Publication 2012
Bacteria Biological Assay Buffers cDNA Library CDTA Clone Cells coelenterazine Deoxyribonucleases furimazine HaloTag hen egg lysozyme HEPES Luminescence Mice, House Mutation N-dodecyl-L-lysine amide Parent Promega Tergitol Thiourea
2
Cell Permeabilization and BRET Assay
Cited 31 times
Cells were washed with DPBS, harvested by trituration, and transferred to opaque black or white 96-well plates containing diluted drug solutions. For assays with nucleotide-free heterotrimers (Fig. 4), cells were washed with permeabilization buffer containing 140 mm KCl, 10 mm NaCl, 1 mm MgCl2, 0.1 mm K-EGTA, 20 mm NaHEPES (pH 7.2), harvested by trituration, and permeabilized in the same buffer containing 10 μg ml−1 high purity digitonin (EMD Millipore, Burlington, MA). Measurements were made from permeabilized cells supplemented either with GDP (0.5 mm) or apyrase (2 units ml−1; Sigma) and agonist. BRET and luminescence measurements were made using a Mithras LB940 photon-counting plate reader (Berthold Technologies GmbH, Bad Wildbad, Germany). Coelenterazine h (5 μm; Nanolight, Pinetop, AZ) or furimazine (Nano-Glo; 1:1000, Promega Corp.) were added to all wells immediately prior to making measurements with Rluc8 and Nluc (or Nluc fragments), respectively. Raw BRET signals were calculated as the emission intensity at 520–545 nm divided by the emission intensity at 475–495 nm. Net BRET was this ratio minus the same ratio measured from cells expressing only the BRET donor. NES–venus–mG fluorescence in Fig. 2 was measured using a Guava 6HT/2L flow cytometer (excitation 488 nm, detection 525/30 nm) and reported as average fluorescence from all positive cells.
Wan Q., Okashah N., Inoue A., Nehmé R., Carpenter B., Tate C.G, & Lambert N.A. (2018). Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells. The Journal of Biological Chemistry, 293(19), 7466-7473.
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Publication 2018
Apyrase Biological Assay Buffers Cells coelenterazine Digitonin Egtazic Acid Fluorescence furimazine Luminescent Measurements Magnesium Chloride Nucleotides Pharmaceutical Solutions Promega Psidium guajava Sodium Chloride Tissue Donors
3
Measuring G Protein Activation via BRET
Cited 17 times
Agonist-dependent cellular measurements of BRET between Venus-Gβ1γ2 and masGRK3ct-Rluc8 or masGRK3ct-Nluc were performed as described previously (80 (link)) to examine the activation of G protein signaling in live cells. Sixteen to twenty-four hours after transfection, HEK 293T/17 cells were washed once with phosphate-buffered saline (PBS) containing 5 mM EDTA (EDTA/PBS), and were detached by incubation in EDTA/PBS at room temperature for 5 min. Cells were harvested by centrifugation at 500g for 5 min and were resuspended in BRET buffer (PBS containing 0.5 mM MgCl2 and 0.1% glucose). Approximately 50,000 to 100,000 cells per well were distributed in 96-well flat-bottomed white microplates (Greiner Bio-One). The Rluc substrate, ViviRen (Promega), was dissolved in ethanol at a final concentration of 20 mM and stored at −20°C. ViviRen was dissolved in BRET buffer immediately before use and added to the cell suspension at a final concentration of 20 µM. The Nluc substrate furimazine was purchased from Promega and used according to the manufacturer’s instructions. BRET measurements were made with a micro plate reader (POLARstar Omega; BMG Labtech) equipped with two emission photomultiplier tubes, which enabled the detection of two emissions simultaneously with the highest possible resolution of 20 ms per data point. All measurements were performed at room temperature. The BRET signal was determined by calculating the ratio of the light emitted by Venus-Gβ1γ2 (535 nm with a 30-nm band path width) to the light emitted by masGRK3ct-Rluc8 or masGRK3ct-Nluc (475 nm with a 30-nm band path width). The average baseline value (basal BRET ratio) recorded before stimulation of cells with agonist was subtracted from the experimental BRET signal values to obtain the ΔBRET ratio.
Masuho I., Ostrovskaya O., Kramer G.M., Jones C.D., Xie K, & Martemyanov K.A. (2015). Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors. Science signaling, 8(405), ra123.
Publication 2015
Buffers Cells Centrifugation Edetic Acid Ethanol furimazine Glucose GTP-Binding Proteins HEK293 Cells Light Magnesium Chloride Phosphates Promega Saline Solution Transfection
4
Binding Assays on Engineered Cells
Cited 9 times
We performed saturation and competition binding assays on stably-transfected cells that we seeded 24 h prior to experimentation in white Thermo Scientific Matrix 96-well microplates. We removed the media from each well and replaced it with HBSS with the required concentration of fluorescent ligand and competing ligand. For pre-incubation experiments with ABEA-X-BY630, we incubated competing unlabeled ligand for 30 min prior to the addition of 250 nM ABEA-X-BY630 for Nluc-A1 expressing cells and 50 nM ABEA-X-BY630 for Nluc-A3 expressing cells. For saturation and competition experiments, upon the addition of fluorescent ligand we incubated cells for 1 h at 37°C (no CO2) and then added the Nluc substrate furimazine (Promega) to a final concentration of 10 μM. For association kinetic experiments on Nluc-A1 expressing cells, we removed media from each well, replaced it with HBSS containing 10 μM furimazine and incubated for 15 min at room temperature in the PHERAstar FS plate reader (BMG Labtech) to allow the signal to reach equilibrium. We then added the required concentration of CA200645, immediately reinserted the plate and read every well once per min for 60 min. For all experiments, we measured the luminescence and resulting BRET using the PHERAstar FS plate reader (BMG Labtech) at room temperature. We again sequentially measured filtered light emissions at 460 nm (80 nm bandpass) and >610 nm (longpass), and calculated the raw BRET ratio by dividing the >610 nm emission by the 460 nm emission.
Stoddart L.A., Johnstone E.K., Wheal A.J., Goulding J., Robers M.B., Machleidt T., Wood K.V., Hill S.J, & Pfleger K.D. (2015). Application of BRET to monitor ligand binding to GPCRs. Nature methods, 12(7), 661-663.
Publication 2015
ABEA-X-BY630 Biological Assay Cells furimazine Hemoglobin, Sickle Ligands Luminescence Motility, Cell Motor Neurons Promega TNFSF14 protein, human
5
Bioluminescent Imaging Protocols
Cited 5 times
In vitro BLI was performed using an IVIS Spectrum one min after addition of Nano-Glo Luciferase Assay Substrate (furimazine; 2-furanylmethyl-deoxy-coelenterazine) following the manufacturer’s specifications (Promega). In vivo bioluminescent imaging was performed on isoflurane-anesthetized animals 5 min after injection of the indicated doses of furimazine either i.p. or i.v. tail-vein. Images were captured with open filter and acquisition times of 60 seconds or less at the indicated settings. Data were analyzed using Living Image software.
Schaub F.X., Reza M.S., Flaveny C.A., Li W., Musicant A.M., Hoxha S., Guo M., Cleveland J.L, & Amelio A.L. (2015). Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis. Cancer research, 75(23), 5023-5033.
Publication 2015
Animals Biological Assay coelenterazine furimazine Isoflurane Luciferases Neoplasm Metastasis oxytocin, 1-desamino-(O-Et-Tyr)(2)- Promega Tail Veins

Most recents protocols related to «Furimazine»

1
Synthetic Cell Encapsulation and NLuc Dynamics
For experiments with NLuc inside synthetic cells, GUVs were generated by encapsulating 20 μL inner solution containing 50 mM Tris-HCl (pH 7.4), 300 mM glucose, 450 nM iLID-6xHis, 100 nM SspB-mCherry, 500 nM NLuc, and 10% v/v OptiPrep. After GUVs were collected, 80 μL of GUV solution was transferred to a 96-well clear flat bottom plate and incubated in the dark for 30 min. Next, 20 μL LCS buffer containing a 20-fold dilution of furimazine stock was added to the well and epifluorescence images of SspB-mCherry were taken immediately. A 100-fold dilution of furimazine stock was added to the well every 15 min for a total of three times during imaging.
For experiments with NLuc attached to the outer membrane of synthetic cells, GUVs were made by encapsulating 20 μL inner solution containing 50 mM Tris-HCl (pH 7.4), 300 mM glucose, 450 nM iLID-6xHis, 100 nM SspB-mCherry, and 10% v/v OptiPrep. After GUVs were collected, 500 nM NLuc was added to the GUV solution and GUVs were incubated at RT for 30 min. Next, 900 μL outer solution was added to the GUV solution and the solution was centrifuged at 2500 g for 10 min at RT. Next, 900 μL of outer solution was removed by gentle pipetting. The GUV pellet was then resuspended by gently pipetting up and down and 80 μL of GUV solution was transferred to a 96 well clear flat bottom plate. Then, 20 μL LCS buffer containing a 20-fold dilution of furimazine stock was added to the well and epifluorescence images of SspB-mCherry were taken immediately. A 100-fold dilution of furimazine stock was added to the well every 15 min for a total of three times during imaging.
Moghimianavval H., Loi K.J., Hwang S.W., Bashirzadeh Y, & Liu A.P. (2024). Light-based juxtacrine signaling between synthetic cells. bioRxiv.
Publication Preprint 2024
2
HiBiT-based Surface Expression Assay
This assay was used to determine relative cell surface expression of transiently transfected β2AR constructs containing N-terminal HiBiT tags (HiBiT-β2ARwt, HiBiT-β2AR_N6A_N15A, HiBiT-β2AR_N6A_N15A_N187A or HiBiT-D113A-β2AR) or pcDNA3.1(+) in HEK293G cells. The 11 amino acid HiBiT tag is a high affinity (KD: 700 pM) peptide that complements the large subunit LgBiT (18.1 kDa) to form the complete nanoluciferase enzyme42 (link). Upon complementation, the complete enzyme becomes active and, in the presence of the furimazine substrate, produces luminescence which can be measured to determine relative HiBiT-tagged protein expression42 (link),66 (link). After 24 h incubation of HEK293G cells expressing HiBiT-tagged β2ARs in 96-well plates at 37 °C and 5% CO2 growth media was aspirated and cells were then incubated with 0.2% purified LgBiT protein and 0.2% Nano-Glo® luciferase substrate furimazine in 100 µl HBSS and incubated at 37 °C for 5 min (to allow for equilibration of LgBiT and the furimazine-based substrate with the HiBiT tagged at the extracellular surface of the receptor) before a single luminescence measurement (open channel, 2000 gain) of each well was taken by a PHERAstar FSX microplate reader. As LgBiT is membrane impermeable, this method provides an estimate of the cell surface expression of the HiBiT-tagged protein67 (link).
Cullum S.A., Platt S., Dale N., Isaac O.C., Wragg E.S., Soave M., Veprintsev D.B., Woolard J., Kilpatrick L.E, & Hill S.J. (2024). Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists. Communications Biology, 7, 417.
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Publication 2024
3
In Vivo Bioluminescent Imaging of laNK92-nLuc Cells
We also used the ARRIVE reporting guidelines.21 (link) First, mice were preconditioned by one time IP injection of 20 mg/kg of busulfan (Millipore Sigma, B2635-25G).22 (link) The next day, 2M laNK92-nLuc cells were injected IP into female hIL-2 NOG aged 4–6 weeks (model# 13 440-F), hIL-15 NOG (model# 13 683-F), and CIEA NOG (model# NOG-F) mice (Taconic Biosciences). One CIEA NOG mouse received daily injection of hIL-2 (50K UI) for five consecutive days, whereas the second one did not receive any hIL-2. Before imaging, mice were anesthetized by 2% isoflurane and then injected IP with furimazine (Promega) (2 µL furimazin in 198 µL of DPBS). The BLI in each mouse was detected within 30 s of furimazine injection with acquisition time of 60 s using an IVIS Lumina III Imaging System. To determine the fold change in mass of laNK92-nLuc cells over time, the total BLI of the mouse body on day 35 was divided by the total BLI of the same mouse on day 1.
Khoshtinat Nikkhoi S., Yang G., Owji H., Grizotte-Lake M., Cohen R.I., Gil Gonzalez L., Massumi M, & Hatefi A. (2024). Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice. Journal for Immunotherapy of Cancer, 12(3), e008295.
Publication 2024
4
In Vivo Bioluminescence Imaging of CAR T Cells and Tumors

Timing: 1 h

This section outlines the procedure for bioluminescence imaging of GBM-bearing mice treated with the traceable CAR T cells using an IVIS (Figure 7A).

Determine the best timing for your in vivo imaging sessions. The initial infiltration of traceable CAR T cells can be observed as early as 1–2 days post-adoptive transfer and can be repeated every two days for weeks.

Calibrate and warm up the IVIS imaging system according to the manufacturer’s instructions.

Set the optimal parameters (exposure time, binning, FOV, and F/stop) for nano-luciferase imaging on the IVIS software.

Anesthetize the mice using an isoflurane/oxygen mixture. Start with 4% isoflurane for induction and then maintain anesthesia with 1–3% isoflurane.

Remove the fur on top of the head of your recipient mice using a trimmer or hair removal cream.

Dilute 5 μL of the furimazine 20× stock solution 20-fold in sterile saline or PBS (100 μL /mouse in total) for injection.

Immediately inject 100 μL of the 1× furimazine solution into the mice retro-orbitally with a 31-gauge insulin syringe.

Wait for 1 min to allow substrate distribution and reaction.

Place the anesthetized mice in the prone position on the imaging platform inside the IVIS chamber.

Note: Ensure that the mice are evenly spaced and not overlapping. Make sure the flow of the isoflurane/oxygen mixture is allowed in the imaging chamber, since the mice need to remain anesthetized during the whole process.

Close the IVIS chamber to ensure darkness and start the imaging acquisition.

Once imaging is complete, remove the mice from the IVIS chamber. Monitor the mice until they fully recover from anesthesia.

Wait for ≥ 60 min to allow the nano-luciferase signal to wean off.

Set the optimal parameters (exposure time, binning, FOV, and F/stop) for firefly luciferase imaging on the IVIS software.

Anesthetize the mice using an isoflurane/oxygen mixture. Start with 4% isoflurane for induction and then maintain anesthesia with 1–3% isoflurane.

Inject 50 μL of the D-luciferin solution into the mice retro-orbitally with a 31-gauge insulin syringe. Avoid using the same eye you have used for the furimazine solution injection.

Wait for 1 min to allow substrate distribution and reaction.

Place the anesthetized mice in the prone position on the imaging platform inside the IVIS chamber. Ensure that the mice are evenly spaced and not overlapping.

Close the IVIS chamber to ensure darkness and start the imaging acquisition. Troubleshooting problem 5.

Once imaging is complete, remove the mice from the IVIS chamber. Monitor the mice until they fully recover from anesthesia.

Return the mice to their home cages and monitor for any adverse reactions, especially at the injection site.

Save the raw and analyzed images in a secure location. Document the imaging parameters, mouse details, and any observations made during the imaging session (Figure 7B).

In vivo bioluminescence imaging of CAR T cells and tumors in mice

(A) Schematic representation of the imaging procedure. Mice injected with CAR T cells expressing nLuc are placed inside the imaging chamber of an IVIS. Furimazine substrate is administered, and after an 1-h waiting period, the mice are again placed inside the imaging chamber, this time having tumors imaged by fLuc. Subsequent administration of D-Luciferin allows for the detection of fLuc-expressing tumors.

(B) Representative bioluminescence images of mice. The left panel shows the distribution of CAR T cells (nLuc) post furimazine injection. The right panel displays the tumor (fLuc) distribution post-D-Luciferin injection.

Zhang D., Krimitza E., Han K., Su R., Xu D.J., Xu J.R., Gong Y, & Fan Y. (2024). Protocol to generate traceable CAR T cells for syngeneic mouse cancer models. STAR Protocols, 5(1), 102898.
Full text: Click here
Publication 2024
5
Rapamycin-Mediated FRB-FKBP Dimerization Assay
To validate rapamycin-mediated FRB-FKBP dimerization in mycobacteria, Msm cells were grown to mid-log phase (OD600 ≈ 0.5) and plated at a final OD600 = 0.1 with 5-fold serial dilutions of rapamycin. The Nano-Glo® 5× reagent (furimazine substrate + buffer; Promega) was prepared and added to each sample. Luminescence was measured in opaque white plates after incubation with linear shaking at 37 °C for 5 min using a Spark 10 M plate reader (Tecan).
Won H.I., Zinga S., Kandror O., Akopian T., Wolf I.D., Schweber J.T., Schmid E.W., Chao M.C., Waldor M., Rubin E.J, & Zhu J. (2024). Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy. Nature Communications, 15, 4065.
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Publication 2024

Top products related to «Furimazine»

1
Furimazine by Promega
Sourced in United States, United Kingdom, France, Germany
Furimazine is a luminescent substrate developed by Promega for use in bioluminescence assays. It is designed to emit light in the presence of specific enzymes, enabling detection and quantification of various biological processes in research applications.
2
Lipofectamine 2000 by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Canada, Japan, France, Italy, Switzerland, Australia, Spain, Belgium, Denmark, Singapore, India, Netherlands, Sweden, New Zealand, Portugal, Poland, Israel, Lithuania, Hong Kong, Argentina, Ireland, Austria, Czechia, Cameroon, Taiwan, Province of China, Morocco
Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
3
Nano glo luciferase assay system by Promega
Sourced in United States, Germany, Japan
The Nano-Glo Luciferase Assay System is a luminescent reporter assay for the detection and quantification of luciferase activity in cell-based assays. The system utilizes a proprietary luciferase enzyme and optimized assay reagents to generate a robust, stable luminescent signal.
4
Nanoluc luciferase assay by Promega
Sourced in Germany
NanoLuc Luciferase Assay is a bioluminescence-based reporter system developed by Promega. It utilizes the NanoLuc luciferase enzyme, which is a small, bright, and stable luciferase that can be used as a reporter in various applications. The assay provides a sensitive and quantitative method for measuring luminescence signals.
5
Mithras lb 940 by Berthold Technologies
Sourced in Germany, United States, France, Switzerland, United Kingdom, Japan
The Mithras LB 940 is a multi-mode microplate reader designed for a wide range of applications. It features a flexible optical system with multiple detection modes, including absorbance, fluorescence, and luminescence. The instrument is capable of performing a variety of assays, such as cell-based, enzymatic, and biochemical analyses.
6
Pherastar fs plate reader by BMG Labtech
Sourced in Germany, United Kingdom, United States, France
The PHERAstar FS is a high-performance, multi-mode microplate reader designed for a wide range of applications. It offers rapid and sensitive detection of fluorescence, luminescence, and absorbance signals in 6- to 384-well microplates.
7
Nano glo by Promega
Sourced in United States
Nano-Glo is a bioluminescent reporter system developed by Promega. It utilizes a genetically engineered luciferase enzyme that emits light upon the addition of a specific substrate. This system can be used to measure various biological activities, such as gene expression, protein-protein interactions, and cell viability.
8
Victor x5 multilabel plate reader by PerkinElmer
Sourced in United States, Singapore
The VICTOR X5 Multilabel Plate Reader is a high-performance microplate reader designed for a variety of applications. It features multiple detection modes, including absorbance, fluorescence, and luminescence. The instrument is capable of reading 6- to 384-well microplates and provides reliable and accurate results.
9
Clariostar by BMG Labtech
Sourced in Germany, United States, United Kingdom, France, Australia, New Caledonia, Japan, Canada
The CLARIOstar is a high-performance microplate reader developed by BMG LABTECH. It is designed to provide accurate and reliable measurement of fluorescence, luminescence, and absorbance in microplate-based assays. The CLARIOstar is capable of reading 96- and 384-well microplates and offers a wide range of detection modes to support a variety of experimental applications.
10
Nano glo luciferase assay substrate by Promega
Sourced in United States
The Nano-Glo Luciferase Assay Substrate is a reagent used to measure the activity of luciferase reporter proteins in cell-based assays. It provides a sensitive and stable luminescent signal that is proportional to the amount of luciferase present in the sample.

More about "Furimazine"

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Researchers can utilize Furimazine to analyze and compare a wide range of protocols from literature, preprints, and patents, including those involving Lipofectamine 2000, Nano-Glo Luciferase Assay System, NanoLuc Luciferase Assay, Mithras LB 940, PHERAstar FS plate reader, Nano-Glo, VICTOR X5 Multilabel Plate Reader, and CLARIOstar.
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