The DPPH• radical scavenging activity of FS and NFS extracts was determined according to Chiellini et al. [40 (link)]. The absorbance was recorded at 517 nm, and the extract concentration corresponding to 50% of DPPH inhibition (EC50) was measured according to Gabriele et al. [41 (link)]. The oxygen radical absorbance capacity (ORAC) of FS and NFS extracts was determined as described by Gabriele et al. [39 ]. AAPH was used as a peroxyl radicals generator and fluorescein as the probe. Fluorescein fluorescence decay was read at λex 485 nm and λem 514 nm using a VictorTM X3 Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA). Results were expressed as ORAC units (µmol TE/g FW) using Trolox as the reference standard. The ferric-reducing antioxidant power (FRAP) assay was used to evaluate the ability of FS and NFS extracts to reduce ferric iron (Fe3+) to ferrous iron (Fe2+) [42 (link)]. The absorbance was measured at 593 nm (Perkin Elmer UV/VIS Lambda 365, Waltham, MA, USA), and results were expressed as Fe2+ equivalents (µM) using a standard curve of FeSO4·7H2O. The Fe2+ chelation ability of FS and NFS extracts was determined as described by Chelucci et al. [42 (link)]. The absorbance was read at 562 nm (Perkin Elmer UV/VIS Lambda 365, Waltham, MA, USA), and results were expressed as EC50 values referring to the extract concentration corresponding to 50% of Fe2+ chelation.
Victortm x3 multilabel plate reader
Manufactured by PerkinElmer
Sourced in United States
The VICTORTM X3 Multilabel Plate Reader is a versatile instrument designed for high-performance detection of a wide range of assay types in microplates. It offers a compact and efficient solution for various research and analytical applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Uv vis lambda 365, by PerkinElmer (1 mentions)
Flsar, by Merck Group (1 mentions)
Atp colorimetric assay kit, by Abcam (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Cyquant direct cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Pore polycarbonate membrane insert, by Corning (1 mentions)
Matrigel, by Corning (1 mentions)
Sorafenib, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Etoposide, by Merck Group (1 mentions)
Sorafenib, by LC Laboratories (1 mentions)
Wst 1 solution, by DoGenBio (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Pkh26 red fluorescent cell linker kit for general cell membrane labeling, by Merck Group (1 mentions)
6 well culture plate, by Corning (1 mentions)
Versene solution, by PanEco (1 mentions)
17 protocols using victortm x3 multilabel plate reader
1
Antioxidant Capacity Evaluation of Fruit Extracts
2
Quantifying Cytoprotective Effects of H2S
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HL-1 cells were seeded in 12-well plates at 10,000 cells per well in complete Claycomb medium. Cells were preconditioned with GYY4137 over 12 hours and ATP content was analyzed periodically. To evaluate the cardio-protective effect of H2S during nutrient deprivation, HL-1 cells were preconditioned overnight with GYY4137 and the culture medium was then replaced with PBS to promote starvation. ATP content was analyzed at various times. To measure ATP, cells were washed twice with PBS buffer and then lysed with the addition of somatic cell ATP releasing reagent (FLSAR, Sigma-Aldrich). The supernatant was collected and kept at −20°C until analysis. ATP content was measured with the bioluminescent ATP somatic cells assay kit (FLASC, Sigma-Aldrich). Luminescence of triplicate samples was read on a VICTORTM X3 Multilabel Plate Reader (PerkinElmer) with a test wavelength of 570 nm. Rat hearts were explanted and stored at −80°C for ATP quantification with the colorimetric ATP Assay Kit (Abcam) following the manufacturer’s instructions. Briefly, tissue samples were homogenized in ATP assay buffer, deproteinized with 1 M perchloric acid, and ATP content was determined by absorbance at 570 nm. ATP levels from experimental samples were compared to sham samples (explanted hearts without treatment).
3
Assessing Migration, Invasion, and Proliferation of Glioma Cells
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After stable overexpression LN229 and U343 and knockdown U87 cells reached ~70% confluence, the cells were scratched with a 200 µL pipette tip, washed with DPBS, and incubated at 37 °C. Wound healing was observed for 12 h and 24 h at the scratched line, and the area around the scratched line was photographed. The invasion assay was performed using an 8.0 μm pore polycarbonate membrane insert (Corning, 353097). Transwell inserts coated with Matrigel (Corning, 354234) were used, and 2 × 104 cells/well were plated in the upper chamber. The lower chamber was filled with 600 μL of serum-free medium. After incubation for 48 h, cells infiltrated with 100% methanol were fixed. After staining with 1% crystal violet (Sigma‒Aldrich, V5265), the infiltrated cells were observed under a microscope. For the colony formation assay, 1 × 103 cells were plated in 6-well plates. After culturing the cells at 37 °C for 2 weeks, colonies formed on each of the three plates were measured. Cell proliferation was assessed according to the manufacturer’s protocol (Invitrogen, C35011) using the CyQuant Direct Cell Proliferation Assay Kit. All experiments were performed in triplicate, and cell proliferation was determined using a VICTORTM X3 Multilabel Plate Reader (PerkinElmer).
4
Cell Viability Assay with Sorafenib and Etoposide
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For the cell viability assays, the cells were seeded in 96-well plates at a density of 5000 cells/well (100 μL total volume/well) and were grown for 24 h. The cells were then washed with sterile phosphate-buffered saline and maintained in low-glucose (5.56 mM) DMEM supplemented with 10% FBS with/without sodium pyruvate (1 mM, Thermo Fisher Scientific, Waltham, MA, USA) for 24 h before adding the sorafenib (LC Laboratories, Woburn, MA, USA) or etoposide (Sigma-Aldrich, Saint Louis, MO, USA). The cells were treated with different concentrations of sorafenib or etoposide, and the controls were treated with vehicle (DMSO, Sigma-Aldrich). After 24-h treatment, WST-1 solution (DoGenBio, Seoul, South Korea) was added to the cells for 1–2 h, and the absorbance at 450 nm was then measured using a VICTORTMX3 Multilabel Plate Reader (PerkinElmer Inc., Waltham, MA, USA).
5
Cytocompatibility Evaluation of NETA-Loaded Biomaterials
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Cytocompatibility of the drug-free and NETA-loaded segments was evaluated (n = 5) using an MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye). For this assay, a cell suspension with density of 2 × 104 cells/well in 100 µL was seeded on 96 well plates and incubated for 24 h at 37 °C and 95% relative humidity containing 5% CO2. Afterward, the growth medium was aspirated and replaced with a growth medium consisting of the extracted leachates, with the cells thereafter grown for an additional 1, 3, 5 and 7 days. Throughout the 7-day testing period, the growth medium containing the extracted leachates was replenished every 2 days. At the end of each designated time point (1, 3, 5, and 7 days), MTT solution (10 µL) was added to each well plate, followed by an addition of 100 µL solubilizing buffer after 4 h, and incubation overnight under the standard culture conditions. The absorbance’s reading of each treatment sample was thereafter taken in triplicate at 570 nm against a 690 nm background using a VICTORTM X3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). In addition to the treatment arms (the NETA-loaded and drug-free segments), NIH/3T3 cells in culture medium and cells in culture medium with DMSO were used as negative and positive controls, respectively.
6
Quantifying Cellular Reactive Oxygen Species
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ROS were quantified using 2′,7′-dichlorofluorescin-diacetate (H2-DCF-DA, Sigma-Aldrich, St. Louis, MO, USA), as previously described [17 (link)]. Upon cleavage of the acetate groups by intracellular esterase and oxidation, the H2−DCF-DA is converted to the fluorescent 2′,7′-dichlorofluorescein (DCF). Briefly, the intestinal cells were seeded into 96-well plates and allowed to adhere overnight. ROS level was measured after the exposure to BOS (1 µg/mL) or to CUR (1 µg/mL) for 24 h. Treatments were removed and H2DCF-DA was added to obtain a final concentration of 50 µM in each well. The plate was incubated for 30 min at 37 °C and washed with phosphate-buffered saline (PBS). DCF fluorescence intensity was measured at excitation 485 nm–emission 535 nm, using VICTORTMX3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA), before and after the addition of 500 µM H2O2 on each well.
7
Assessing Adenoviral Transduction in Murine Cells
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Human cells were seeded in 96-well plates (1 × 104 cells/well) and incubated overnight at 37 °C and 5% CO2. Fresh serum from naïve C57BL/6 mice, Rag2-/-, or Ighmtm1Che C57BL/6 mice was incubated with 1 × 109 vp of vector (HAdV-5 luc) in a final volume of 50 μL (30 min at 37 °C). The potential interactions with mouse FX were inhibited by the addition of 40 μg/mL X-bp. Samples were then diluted 200-fold in serum-free medium. A total of 1000 vp/cell were added onto cells for 2 h at 37 °C, then media was replaced with fresh media containing 2% FBS, and after further ~16 h at 37 °C cells were rinsed with PBS and harvested by freezing and thawing in Reporter Lysis Buffer (Promega, UK) for both luciferase assay (Promega, Southampton, UK) and protein content measurement (BCA assay) using a VICTORTM X3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA).
8
Labeling and Harvesting Cell Sheets
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Prior to cell sheet formation MSC (passage 2–4) were labeled by PKH26 Red dye (PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling, Sigma-Aldrich, Milwaukee, WI, USA). The cells were detached with 0.05% trypsin, labeled and seeded at 3 × 106/well density in a 6-well culture plate (Corning, Corning, NY, USA). Forming CSs were maintained in DMEM, 10% FBS, penicillin/streptomycin for at least 48 h prior to experiments. The transgene expression was verified by enzyme-linked immunosorbent assay to assess SCF concentration in conditioned media. The Mouse SCF ELISA Kit (ab100740, Abcam, Cambridge, MA, USA) was used. An optical density was measured using VictorTM X3 Multi Label Plate Reader (Perkin Elmer Inc, Waltham, MA, USA). The cell sheet was harvested by incubation in Versene solution (Paneco, Moscow, Russia) until it self-detached within 5–7 min.
9
Antioxidant Activity of HaCaT Cells
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The human keratinocyte cell line HaCaT was cultured at 37 °C in a humidified incubator with 5% CO2 in Dulbecco’s Medium (DMEM) supplemented with 10% foetal bovine serum, two mM L-glutamine, 50 U/mL penicillin, and 50 g/mL streptomycin. To assess the intracellular antioxidant activity of HaCaT cells, they were seeded at a density of 3104 cells per well in 96-well plates. The studies were conducted 24 h after incubation at 37 °C in 5% CO2.
Cell viability was determined by decreasing MTT as described in our previous work [30 (link)]. At 37 °C in 5% CO2, HaCaT cells were treated for 24 h with various concentrations of extracts (0.160–5 mg/mL). The treatment medium was changed to MTT in HBSS (0.5 mg/mL) for two hours at 37 °C in 5% CO2. Formazan crystals were dissolved in isopropanol after being washed with HBSS. The formazan concentration was determined (570 nm, reference filter 690 nm) using the VICTORTMX3 multilabel plate reader (PerkinElmer, Waltham, MA, USA). The viability of the cells was expressed as a percentage of total vitality.
Cell viability was determined by decreasing MTT as described in our previous work [30 (link)]. At 37 °C in 5% CO2, HaCaT cells were treated for 24 h with various concentrations of extracts (0.160–5 mg/mL). The treatment medium was changed to MTT in HBSS (0.5 mg/mL) for two hours at 37 °C in 5% CO2. Formazan crystals were dissolved in isopropanol after being washed with HBSS. The formazan concentration was determined (570 nm, reference filter 690 nm) using the VICTORTMX3 multilabel plate reader (PerkinElmer, Waltham, MA, USA). The viability of the cells was expressed as a percentage of total vitality.
10
Quantifying Intestinal Antibody Responses
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Secreted antibodies in the intestinal lavage were analysed using the Mouse Immunoglobulin Isotyping ELISA Kit (BD Biosciences, Heidelberg, Germany) applied as recommended by manufacturer. For the EcN specific ELISA a 96-well plate was coated with heat inactivated EcN (1:10). EcN was fixed with 0.025% glutaraldehyde for 10 min at room temperature (RT) followed by short washing with distilled water. The plate was blocked with 5% BSA in PBST (used as a sample diluent as well) for 1 h at RT. Between each step the plate was washed 4-5 times with PBST. Samples (1:20) were mounted on the plate for 2 h at 37 °C. For detection AB Biotin Rat Anti Mouse IgA (1:200), IgM (1:200) and IgG2a (1:125) were used (BD Pharmingen). Antibodies were incubated for 1 h at 37 °C. Subsequently Streptavidin HRP Enzyme (1:1000; BD Pharmingen) was added and incubated for 1 h at RT in the dark. As a substrate for the enzyme TMB Substrate Reagent Set (1:2; BD Pharmingen) was used for 10–20 min at RT in the dark. The reaction was stopped using sulfuric acid (1 M). The absorbance was read at 450 nm in the VICTORTM X3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA).
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