Botulinum neurotoxin serotype A (holotoxin and toxin complex) were obtained from Metabiologics at 1 mg/mL (Madison, WI, USA). CANARY® Zephyr BoNT/A kit (25 reactions) containing assay buffer, reconstitution buffer, negative control, positive control, B-cell biosensors, microcentrifuge tubes, and immunomagnetic capture beads were obtained from PathSensors, Inc. (Baltimore, MD, USA). All reagents from the kit were stored at 4 °C while the biosensors are stored in liquid nitrogen. The CANARY® detection system consists of a laptop, a small microcentrifuge (SCILOGEX D1008, Rocky Hill, CT, USA), and a luminometer (Sirius L Tube Luminometer, TITERTEK-Berthold, Pforzheim, Germany). Whole milk, 2% milk, non-fat milk, orange juice (no pulp), carrot juice, apple juice, green bean baby food, 80:20% ground beef, and smoked salmon were bought from a local supermarket.
Sirius l tube luminometer
Manufactured by Berthold Technologies
Sourced in Germany
The Sirius L Tube Luminometer is a laboratory instrument designed to measure luminescence. It is capable of detecting and quantifying light emission from various types of samples, such as those containing luciferase reporter enzymes or bioluminescent organisms. The Sirius L Tube Luminometer provides accurate and reliable luminescence measurements to support research and analysis in a range of scientific applications.
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Lab products found in correlation
D1008, by Scilogex (1 mentions)
Polybrene transfection reagent, by Merck Group (1 mentions)
Dual luciferase reporter assay protocol, by Promega (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Pierce gaussia luciferase flash assay kit, by Thermo Fisher Scientific (1 mentions)
Britelite plus reporter gene assay system, by PerkinElmer (1 mentions)
Synergy neo2 microplate reader, by Agilent Technologies (1 mentions)
8 protocols using sirius l tube luminometer
1
Rapid Detection of Botulinum Neurotoxin A
2
3'UTR Fgf10 Luciferase Assay
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A psiCHECK−2 vector and a Dual-Luciferase Reporter Assay System (C8021, E1910, Promega) were used for target validation. Two ~700 bp long sequences of the 3′UTR of Fgf10 mRNA that contained either miR-327 binding site 1 (bs1) or bs2 were separately cloned into a psiCHECK-2 vector. HEK293T human embryonic kidney cells (ATCC) were transfected with a transfection medium containing the recombinant plasmids (1 µg µL−1 DNA) mixed with Polybrene transfection reagent (1 mg mL−1, Sigma-Aldrich) in OPTI-MEM medium (Thermo Fisher Scientific Inc.) for 16 h. Cells were further transfected with miR-327 mimics using DharmaFECT siRNA transfection reagent. Renilla and Firefly luciferase signals were measured 48 h after the initial transfection using the Dual-Luciferase Reporter Assay Protocol (Promega) and a Sirius L Tube Luminometer (Titertek-Berthold). To mutate the binding sites of miR-327 in the 3′UTR of Fgf10 mRNA, PCR amplification with primers containing 5 mismatches in the miR-327 bs1 or bs2 were used and the mutated recombinant plasmids were inserted into the psiCHECK-2 vector for further luciferase quantification. All primers used are listed in Supplementary Table 1 .
3
Plasma Treatment Effects on Soybean Sprouts
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Soybean seeds without plasma treatment (control) and those treated with 22.1 kV for 12 s were grown for 6 days. Homogenization of soybean sprout was performed in 1 ml ice-cold phosphate buffer saline (0.05 mol/l, pH 7.4), centrifuged (12,000 × g) for 10 min at 4 °C, and supernatant was collected to detect soluble protein concentration using a bicinchoninic acid protein assay kit (Sigma-Aldrich, St. Louis, MO, USA). Activities of MDA, SOD, POD, and CAT were analyzed using kits from Sigma-Aldrich, according to the manufacturer’s instructions. On day 6, soybean sprouts were collected and homogenized in 1× ATP reaction buffer. After centrifugation (12,000 × g, 5 min, 4 °C), ATP concentration in the supernatant was determined using an ATP determination kit (Invitrogen, Carlsbad, CA, USA). Relative light unit values were measured using a luminometer (Sirius L Tube Luminometer, Titertek Berthold, Bleichstraße, Pforzheim, Germany).
4
Mitochondrial ATP Synthesis Measurement
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The rate of mitochondrial ATP synthesis driven by CI, CII, and CIII was measured in digitonin-permeabilized cells. After trypsinization, cells (10 × 106 mL−1) were suspended in a buffer containing 150 mM KCl, 25 mM Tris–HCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 0.1% bovine serum albumin, 10 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4, kept at room temperature for 15 min, then incubated with 50 μg mL−1 digitonin until 90–100% of cells were positive to Trypan Blue staining. Aliquots of 3 × 105 permeabilized cells were incubated in the same buffer in the presence of the adenylate kinase inhibitor P1,P5-di(adenosine-5′) pentaphosphate (0.1 mM) and OXPHOS complexes substrates, chemiluminescence was determined as a function of time with Sirius L Tube luminometer (Titertek-Berthold, Pforzheim, Germany). The chemiluminescence signal was calibrated with an internal ATP standard after the addition of 10 μM oligomycin. The rates of the ATP synthesis were normalized to protein content and citrate synthase (CS) activity49 (link).
5
Measuring Mitochondrial ATP Synthesis
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The rate of mitochondrial ATP synthesis was measured in digitonin-permeabilized cells as previously described [40 (link)]. Briefly, after trypsinization, cells (10 × 106/mL) were suspended in a buffer containing 150 mM KCl, 25 mM Tris-HCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 0.1% bovine serum albumin, 10 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4, kept at room temperature for 15 min, then incubated with 50 μg/mL digitonin until 90–100% of cells were positive to Trypan Blue staining. Aliquots of 3 × 105 permeabilized cells were incubated in the same buffer in the presence of the adenylate kinase inhibitor P1,P5-di(adenosine-5′) pentaphosphate (0.1 mM) and CI substrates 1 mM malate plus 1 mM pyruvate. After the addition of 0.2 mM ADP, chemiluminescence was measured during time (Sirius L Tube luminometer, Titertek-Berthold, Pforzheim, Germany). The chemiluminescence signal was calibrated with an internal ATP standard after the addition of 10 μM oligomycin. The rates of the ATP synthesis were normalized to protein content and CS activity.
6
Quantifying CI-driven ATP Synthesis
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The rate of CI-driven ATP synthesis was measured in digitonin-permeabilized cells treated or not with 50 μg/mL chloramphenicol. Cells were harvested by trypsinization, pellets were washed in PBS and the cells (10 × 106 /mL) were suspended in a buffer containing 150 mM KCl, 25 mM Tris-HCl, 2 mM EDTA, 0.1% BSA, 10 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4. Then, cellular suspensions were incubated with 50 μg/mL digitonin for 1 minute at RT with gentle agitation. As a result of the permeabilization 90%–100% of cells were positive to Trypan Blue staining. Aliquots of 3 × 105 permeabilized cells were incubated in the same buffer with 0.9 mM Na+ pyruvate, 0.9 mM K+ malate, and 10 μL of ATP assay mix (Sigma-Aldrich; #FLAAM) in the presence of 0.1 mM adenylate kinase inhibitor P1,P5-di(adenosine-5′) pentaphosphate. After the addition of 0.09 mM ADP, chemiluminescence was determined as a function of time with Sirius L Tube luminometer (Titertek-Berthold, Pforzheim, Germany). The chemiluminescence signal was calibrated with 11 μM internal ATP standard (Sigma-Aldrich; #FLAAS) after the addition of 1 μM rotenone The rates of the ATP synthesis were normalized to protein content and citrate synthase (CS) activity.
7
Quantifying Viral Luciferase Activity
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To determine the activity of Gluc, 50 μL of viral culture medium or lung tissue homogenate (appropriate dilution applied to avoid over range) were mixed with 50 μL of luciferase substrate using Pierce Gaussia Luciferase Flash Assay Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. The luminescence was detected immediately using Sirius L Tube Luminometer (Berthold, Germany).
Fluc assays were performed using a Britelite plus Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. In brief, MDCK cells growing in 96 well plates were infected by indicated viruses at an MOI of 0.01. After 1 h incubation at 37°C, cells were washed and fresh Opti-MEM containing 2 μg/ml TPCK–trypsin were added. At 24 h post infection (p.i.), the culture medium was discarded, followed by sequentially adding 50 μL PBS and 50 μL substrate. After incubation for 10min, the luminescence was detected immediately using BioTek SYNERGY neo2 Microplate Reader (BioTek, Winooski, VT, USA).
Fluc assays were performed using a Britelite plus Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. In brief, MDCK cells growing in 96 well plates were infected by indicated viruses at an MOI of 0.01. After 1 h incubation at 37°C, cells were washed and fresh Opti-MEM containing 2 μg/ml TPCK–trypsin were added. At 24 h post infection (p.i.), the culture medium was discarded, followed by sequentially adding 50 μL PBS and 50 μL substrate. After incubation for 10min, the luminescence was detected immediately using BioTek SYNERGY neo2 Microplate Reader (BioTek, Winooski, VT, USA).
8
Measuring Mitochondrial ATP Synthesis
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The rate of mitochondrial ATP synthesis driven by CI, CII, and CIII was measured in digitoninpermeabilized cells treated or not with 50 μg/mL chloramphenicol as previously detailed (Ghelli et al, 2013) . Briefly, cells (10 × 10 6 /mL) were suspended in a buffer containing 150 mM KCl, 25 mM Tris-HCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 0.1% bovine serum albumin, 10 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4, and incubated with 50 μg/mL digitonin until 90-100% of cells were positive to Trypan Blue staining. Aliquots of 3 × 10 5 permeabilized cells were incubated in the same buffer in the presence of the adenylyl kinase inhibitor P1,P5-di(adenosine-5′) pentaphosphate (0.1 mM) and OXPHOS complexes substrates, chemiluminescence was determined as a function of time with Sirius L Tube luminometer (Titertek-Berthold, Pforzheim, Germany).
The chemiluminescence signal was calibrated with 11 µM internal ATP standard after the addition of 1 μM oligomycin. 1 μM rotenone was added instead of oligomycin to determine the specific CIdriven ATP synthesis. The rates of the ATP synthesis were normalized to protein content and citrate synthase (CS) activity.
The chemiluminescence signal was calibrated with 11 µM internal ATP standard after the addition of 1 μM oligomycin. 1 μM rotenone was added instead of oligomycin to determine the specific CIdriven ATP synthesis. The rates of the ATP synthesis were normalized to protein content and citrate synthase (CS) activity.
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