Unless specified otherwise, all the used compounds were specified at Table S1 , while cell culture disposables were provided by Sarstedt (Blizne Łaszczyńskiego Poland). Unless specified otherwise, spectrophotometric assays were run either using Ultraspec 3100 Pro (Amersham Biosciences, Amersham, UK) or, for multiple well plate-based assays, Victor 3, 1420 Multilabel Counter (Perkin Elmer, Warsaw, Poland).
Ultraspec 3100 pro
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The Ultraspec 3100 Pro is a high-performance UV/Visible spectrophotometer designed for laboratory applications. It offers accurate and reliable measurements of absorbance, transmittance, and concentration of samples across a wide wavelength range.
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11 protocols using ultraspec 3100 pro
1
Antioxidant Compound Evaluation Protocol
2
Hemolytic Capacity Assay of E. coli
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The hemolytic capacity of the E. coli strains was determined as described [19 (link)] using New Zealand White rabbit erythrocytes (Gabinete Veterinario, Universidad Autónoma de Madrid). Blood was treated with a final concentration of 0.1% (w/v) EDTA pH 7.5 to prevent coagulation. Erythrocytes from 5 ml of blood were collected by centrifugation (3500×g, 15 min, room temperature [RT]), and washed three times with one volume of 0.9% NaCl with slow centrifugation (1000×g, 10 min, RT) between washes. The erythrocyte suspension was diluted to 4% in DMEM, and 0.5 ml of this suspension was mixed with 0.5 ml of induced bacterial culture (OD600 0.4). The mix was centrifuged (2500×g, 1 min, RT) to induce contact of the bacteria with the erythrocytes and then incubated at 37 °C with 5% CO2 during 4 h. The pellet was then gently resuspended and centrifuged (12,000×g, 1 min, RT), and hemoglobin released into the supernatant was measured at 450 nm (OD450) in a spectrophotometer (Ultraspec 3100 pro, Amersham Biosciences). The background average OD450 value obtained with the control strain EcM1 was subtracted from the values of all other samples, and the value of SIEC was taken as reference (100%).
3
Anthocyanin and Flavonol Profiling in Petunia
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Anthocyanin and flavonol absorbances were measured in the P. axillaris × P. exserta F7 RIL population using the same protocol as described by Sheehan et al. (2016) . For each plant, five flowers were phenotyped. For each flower, a disk 8 mm in diameter of petal limb tissue was sampled and placed into 1 mL of MAW extraction buffer (2:1:7 methanol:acetic acid:water). After 48 h in the dark, absorption spectra were measured on a spectrophotometer (Ultraspec 3100 pro, Amersham Biosciences, England, UK). Anthocyanin values represent summed absorbance values over 445–595 nm and flavonol values represent summed absorbance values over 315–378 nm.
Anthocyanins and flavonols were evaluated for the identification of QTLs based on their phenotypic distribution using the “scanone” function in R/qtl v1.46-2 (Broman et al., 2003 (link)). Anthocyanins were normally distributed and analyzed with the “hk” Haley–Knott regression method. Flavonols showed a bimodal distribution reflecting groups with high and low concentrations and were analyzed using the “2part” method. Genome-wide significance level was established using 10,000 permutations using the “n.perm” argument for each of the anthocyanin and flavonol phenotypes at α = 0.05. The R/qtl script is made available onhttps://github.com/Kuhlemeier-lab/Exserta-red/.
Anthocyanins and flavonols were evaluated for the identification of QTLs based on their phenotypic distribution using the “scanone” function in R/qtl v1.46-2 (Broman et al., 2003 (link)). Anthocyanins were normally distributed and analyzed with the “hk” Haley–Knott regression method. Flavonols showed a bimodal distribution reflecting groups with high and low concentrations and were analyzed using the “2part” method. Genome-wide significance level was established using 10,000 permutations using the “n.perm” argument for each of the anthocyanin and flavonol phenotypes at α = 0.05. The R/qtl script is made available on
4
Photometric Assay for Laccase Activity
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Laccase activity was photometrically measured (Ultraspec 3100pro, Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy). The reaction was performed in 100 mM sodium acetate pH 4.5, 2 mM ABTS and an appropriate amount of enzyme solution in a final volume of 1 mL. The oxidation of ABTS was determined after 10 min by photometric assay at 420 nm (ε420 = 36,000 M−1·cm−1). One unit was defined as the amount of enzyme which oxidized 1 μmol of substrate per min [51 (link)].
5
Spectrophotometric Assays and Compound Sourcing
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Unless otherwise specified, all used compounds were specified at Supplement 1 , while cell culture disposables were provided by Sarstedt (Blizne Łaszczyńskiego, Poland). Unless otherwise specified, spectrophotometric assays were run either using Ultraspec 3100 Pro (Amersham Biosciences, Warsaw, Polnad) or, for multiple well plate-based assays, Victor3, 1420 Multilabel Counter (Perkin Elmer, Warsaw, Poland).
6
Enzymatic Activity Assay Using ABTS
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The enzymatic activity was assessed by the ABTS colorimetric assay using a spectrophotometer (Ultraspec 3100pro, Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy). [7 (link)]. The reaction was performed in 100 mM sodium acetate pH 4.5, 5 mM ABTS and an appropriate amount of enzyme solution in a final volume of 1 mL. The oxidation of ABTS was determined after 10 min at 420 nm (ε420 = 36,000 M−1cm−1). One unit was defined as the amount of enzyme which oxidized 1 µmol of substrate per min.
7
Comprehensive Spectrophotometric Assay Protocol
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Unless specified otherwise, all the used compounds were specified in Table S1 , while cell culture disposables were provided by Sarstedt (Blizne, Łaszczyńskiego, Poland). Unless specified otherwise, spectrophotometric assays were run either using an Ultraspec 3100 Pro (Amersham Biosciences, Amersham, UK) or, for multiple well plates-based assays, a Victor 3, 1420 Multilabel Counter (Perkin Elmer, Warsaw, Poland).
8
Erythrocyte Hemolysis Assay for EPEC
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The protocol was
performed as previously described.33 (link) Briefly,
erythrocytes were obtained from a 5 mL blood sample obtained from
New Zealand White rabbits (Granja San Bernardo, Navarra, Spain). The
blood was treated adding 500 μL of EDTA 1% (w/v) pH 7.5 (100
μL for each ml) to avoid coagulation and was centrifuged (3500g, 15 min, RT) to concentrate the erythrocytes. The solution
was washed 3 times with one volume of NaCl 0.9% (w/v) followed by
centrifugation (1000g, 10 min, RT). The assembly
of the T3SS was induced in the indicated cultures as described above
until they reached OD600 0.4. Then, 0.5 mL of the cultures
and 0.5 mL of the erythrocytes suspension (previously diluted to a
4% in DMEM) were mixed in 1.5 mL tubes. The final mixture was centrifuged
(2500g, 1 min, RT) to induce the contact of the bacteria
with the erythrocytes, incubated at 37 °C with 5% CO2 during 4 h, and then the erythrocyte pellets were softly resuspended.
The samples were centrifuged (12000g, 1 min, RT)
and the hemoglobin release to the supernatant was measured at OD450 in a spectrophotometer (Ultraspec 3100 pro, Amersham Biosciences).
The hemolysis induced by the wt EPEC strain was considered 100%. Background
hemolysis induced by E. coli K-12 strain EcM1 was
subtracted from the values obtained in all samples. The experiment
was repeated three times, each one with triplicates.
performed as previously described.33 (link) Briefly,
erythrocytes were obtained from a 5 mL blood sample obtained from
New Zealand White rabbits (Granja San Bernardo, Navarra, Spain). The
blood was treated adding 500 μL of EDTA 1% (w/v) pH 7.5 (100
μL for each ml) to avoid coagulation and was centrifuged (3500g, 15 min, RT) to concentrate the erythrocytes. The solution
was washed 3 times with one volume of NaCl 0.9% (w/v) followed by
centrifugation (1000g, 10 min, RT). The assembly
of the T3SS was induced in the indicated cultures as described above
until they reached OD600 0.4. Then, 0.5 mL of the cultures
and 0.5 mL of the erythrocytes suspension (previously diluted to a
4% in DMEM) were mixed in 1.5 mL tubes. The final mixture was centrifuged
(2500g, 1 min, RT) to induce the contact of the bacteria
with the erythrocytes, incubated at 37 °C with 5% CO2 during 4 h, and then the erythrocyte pellets were softly resuspended.
The samples were centrifuged (12000g, 1 min, RT)
and the hemoglobin release to the supernatant was measured at OD450 in a spectrophotometer (Ultraspec 3100 pro, Amersham Biosciences).
The hemolysis induced by the wt EPEC strain was considered 100%. Background
hemolysis induced by E. coli K-12 strain EcM1 was
subtracted from the values obtained in all samples. The experiment
was repeated three times, each one with triplicates.
9
Roselle Fruit Juice Phenolic Content
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Total phenolic content of Roselle fruit juice was determined according to the Folin-Ciocalteu procedure that was adopted from Youssef et al. (2015) . Folin-Ciocalteu reagent (10%) with 1.5 mL was mixed with 1.2 mL of 7.5% sodium carbonate, Na 2 CO 3 solution, then 0.3 mL of Roselle juice sample was added. The test tubes were allowed to stand for one hour at ambient temperature, and the absorption was measured 765 nm using a spectrophotometer (Ultraspec 3100 Pro, Amersham Pharmacia Biotech, UK) against a blank. All the experiments were carried out in triplicate. TPC was obtained from the calibration curve prepared with gallic acid at concentrations of 0, 50, 100, 150, 250 and 500 mg/L.
10
Quantifying Pigment Levels in Frozen Florets
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Five grams of frozen florets was mixed with 20 mL of methanol and incubated overnight in darkness at room temperature (20 °C). Samples were centrifuged at 20,200×g for 15 min. Absorbance was measured in the supernatant by a spectrophotometric assay (Ultraspec 3100pro, Amersham Pharmacia Biotech Italia -Cologno Monzese, Italy) at 653 and 666 for chlorophyll a and b, respectively, and at 470 nm for carotenoids. Chlorophyll a and b, total chlorophyll and carotenoid contents were calculated according to Wellburn (1994) .
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