Chondrocytes were stimulated as indicated above and supernatants were harvested and centrifuged. For matrix metalloproteinase (MMP) activity determination, supernatants were incubated with p-aminophenylmercuric acetate for 12 h at 37ºC to activate MMPs. Then, supernatants were transferred to a 96-well plate. After addition of the 5-FAM peptide substrate (AnaSpec Inc., San Jose, CA, USA), fluorescence was measured at 490 nm (excitation)/520 nm (emission) in a Victor3 microplate reader (PerkinElmer España). Prostaglandin E 2 (PGE 2 ) was quantitated in supernatants by radioimmunoassay [23] and nitric oxide (NO) production was assessed by fluorometric determination of nitrite levels [24] using a Victor3 microplate reader (PerkinElmer España).
Victor3 microplate reader
Manufactured by PerkinElmer
Sourced in United States, Spain, Finland
The Victor3 microplate reader is a versatile instrument designed for high-throughput detection and analysis of samples in microplate format. It offers a range of detection modes, including absorbance, fluorescence, and luminescence, allowing users to conduct various assays and experiments. The Victor3 provides reliable and accurate measurements, making it a valuable tool for researchers and laboratories.
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Lab products found in correlation
Hrp conjugated streptavidin, by BD (3 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Proteinase k, by Merck Group (2 mentions)
Prism 6, by GraphPad (2 mentions)
Opteia mouse ige elisa kit, by BD (2 mentions)
Elisa plate, by Thermo Fisher Scientific (2 mentions)
Dmem without phenol red, by Merck Group (1 mentions)
Protein carbonyl colorimetric assay kit, by Cayman Chemical (1 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
Metaphosphoric acid, by Merck Group (1 mentions)
2 vinylpyridine, by Merck Group (1 mentions)
Glutathione assay kit, by Cayman Chemical (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Rf 5301pc spectrofluorophotometer, by Shimadzu (1 mentions)
Cellular ros detection assay kit, by Abcam (1 mentions)
Victor3 multilabel plate reader, by PerkinElmer (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Quantikine sets, by R&D Systems (1 mentions)
84 protocols using victor3 microplate reader
1
Measuring Chondrocyte MMP, PGE2, and NO
2
Piezoelectric Stimulation Enhances MSC Proliferation
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The influence of piezoelectric stimulation on MSCs proliferation was assessed by analysing cell metabolic activity on days 2, 7, 14 and 21 under static (no applied stimuli) and dynamic (cell culture under magnetic stimulation) conditions. An alternating magnetic field (0–230 Oe) was provided using a homemade magnetic bioreactor placed inside the incubator, applying a 0.3 Hz frequency and a 10 mm magnet displacement under the 48-well plate [61 (link)]. The stimulation program was divided into an active period of 16 h based on 5 min of magnetic stimulation and 25 min of resting time, followed by a non-active period of 8 h when no magnetic stimulation was applied [22 (link),62 (link)]. A diagram of the magnetic stimulation program can be found in Scheme 1 .
At different time points, hydrogels were transferred to a new culture plate. The basal medium was replaced for DMEM without phenol red (Sigma-Aldrich) containing the tetrazolium salt MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Biovision) at a working dilution of 1:10. Hydrogels were incubated for 1 h at 37 °C. After that, the supernatant was transferred to a new plate and absorbance at 490 nm was read with a Victor3 microplate reader (PerkinElmer). Gelatin hydrogels without microspheres stimulated (S) and non-stimulated (NS) were used as controls.
At different time points, hydrogels were transferred to a new culture plate. The basal medium was replaced for DMEM without phenol red (Sigma-Aldrich) containing the tetrazolium salt MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Biovision) at a working dilution of 1:10. Hydrogels were incubated for 1 h at 37 °C. After that, the supernatant was transferred to a new plate and absorbance at 490 nm was read with a Victor3 microplate reader (PerkinElmer). Gelatin hydrogels without microspheres stimulated (S) and non-stimulated (NS) were used as controls.
3
SIRT1 Activity Assay Protocol
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Control and treated cells were lysed (20 mM Tris-Cl, pH 7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Nonidet P-40) and proteins were immunoprecipitated with anti-SIRT1-conjugated protein A agarose beads (cat. ab7343 and ab193254, respectively, Abcam, Cambridge, UK). Briefly, the agarose beads were mixed with primary antibody for 4 h at 4 °C (15 µg of anti-SIRT1 antibody/mL of beads). The mixture was then centrifuged and washed with the aforementioned lysis buffer several times. After the last wash, cell lysates (2 mg of total proteins) were incubated overnight with the antibody-beads mixture. The immunecomplexes were centrifuged and washed, and the immunoprecipitates were directly used for the SIRT1 activity assay kit (cat. ab156065, Abcam, Cambridge, UK), according to supplier’s instructions [16 (link),41 (link)]. The reaction mixtures were prepared by mixing 17 µL ddH2O, 5 µL fluoro-substrate peptide, 5 µL NAD+, 5 µl developer, and 13 µL test samples. Fluorescence (ex. 355 nm, em. 460 nm) was detected for 70 min, with 2 min intervals, in a Victor3 microplate reader (PerkinElmer, Waltham, MA, USA). The assays were performed with three replicates.
4
Osteoblast PGE2 Response to Extracellular Vesicles
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Osteoblasts were stimulated with IL-1β (10 ng/mL) in the presence or absence of MV (3.6 × 107 particles/mL), EX (7.2 × 107 particles/mL), or CM (1 mL) for 24 h. Supernatants were used to measure prostaglandin E2 (PGE2) by radioimmunoassay as previously described [27 (link)] using a Victor3 microplate reader (PerkinElmer España, Madrid, Spain).
5
Cell Viability Assay with ATP Measurement
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Cells were added to 96-well plates and treated as above with the concentrations of drugs indicated in the text. After 24 hours, CTG assay reagent (Promega) was diluted 5-fold in PBS, and 80 μL of the diluted reagent was added to each well. Plates were agitated for 2 min and then incubated at rest for 10 min. Total luminescence of each well, corresponding to ATP content, was recorded using a VICTOR3 microplate reader (Perkin Elmer).
6
Protein Carbonyl Colorimetric Assay
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In order to detect the carbonyl content in our samples, a 2,4-dinitrophenylhydrazine (DNPH)-based Protein Carbonyl Colorimetric Assay Kit (cat. 10005020; Cayman Chemical Company, Ann Arbor, MI, USA) was used59 (link). Briefly, control and ELF-MF-exposed cells were harvested and homogenized in cold EDTA-containing phosphate buffer (pH 6.7) (2 × 107 cells/ml). Samples were centrifuged at 16,000 × g for 30 minutes at 4 °C. Supernatants were treated with 1% (w/v) streptomycin sulfate (cat. sc-202821; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) to remove contaminant nucleic acids, as recommended by the manufacturer. After the reaction with DNPH, samples were transferred into 96-well plate, and the formation of the corresponding hydrazones was recorded at 370 nm (extinction coefficient: 0.022 µM−1 × cm−1) by using a Victor3 microplate reader (PerkinElmer Inc.). As the several washing steps may easily cause protein loss, protein levels were determined on the post-washing final pellets, as recommended by the manufacturer. Five independent experiments were carried out.
7
Apoptosis Measurement in DLBCL Cells
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Apoptosis was assessed using the luminescence-based Caspase-Glo 3/7 assay kit (Promega) according to manufacturers' instructions. Exponentially growing DLBCL cell lines were seeded into 96-well plates and incubated in the presence of previously determined saturating concentrations of IMGN529 or rituximab, both alone and in combination, for 24 hours. Caspase-Glo 3/7 reagent was added (50% v/v) to the cells, and plates were incubated for 30 minutes before luminescence detection in a Victor3 microplate reader (PerkinElmer). Additional caspase3/7 activation studies were performed combining IMGN529 with ofatumumab, obinutuzumab, K7153A, and IgG1-SMCC-DM1 in the U-2932 and SU-DHL-4 cell lines using the same protocol.
8
Antiviral Effects of Volatile Oils
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Influenza H1N1 (A/WSN/33) virus–infected Madin-Darby canine kidney epithelial (MDCK) cell (Sethy et al., 2019 (link)) and enterovirus D68–infected rhabdomyosarcoma (RD) cell (Hsieh et al., 2020 (link)) experimental models were used to assess the cytopathic effects of the samples. Briefly, MDCK or RD cells (96-well plate, 2 × 104 per well) were incubated in E10 medium at 5% CO2 for 16–24 h at 37°C and then washed once with Dulbecco’s phosphate-buffered saline before the infection step. The respective cells were infected with influenza or enterovirus at a ninefold median tissue culture infective dose, with or without the addition of the samples. The cells were treated with the volatile oil samples (50 µg/ml) for 72 h at 37°C. The cells were then fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature and were stained using 0.1% crystal violet for 20 min. The optical density of the cells was measured with a VICTOR3™ microplate reader (PerkinElmer).
9
Quantifying Cellular Glutathione Levels
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Total glutathione (tGSH) and glutathione disulfide (GSSG) levels were determined by using a glutathione assay kit (cat. 703002, Cayman Chemical) [53 (link)]. Briefly, control and treated cells were lysed in the MES buffer provided by the kit (1.5 × 107 cells/mL), and centrifuged at
10,000× g for 15 min, at 4 °C. Cell lysates were deproteinized with 5% (w/v) metaphosphoric acid (cat. 239275, Sigma-Aldrich, Milan, Italy) and centrifuged at 4000× g for 5 min, as recommended by the supplier. In order to evaluate the abundance of GSSG, GSH in the samples was first derivatized with 2-vinylpyridine (cat. 132292, Sigma-Aldrich, Milan, Italy). Protein-free samples (50 µL) and the assay cocktail (150 µL) were loaded in a 96-well plate, with three replicates. Absorbance at 405 nm was followed for 30 min, with 5 min intervals, by using a Victor3 microplate reader (PerkinElmer, Waltham, MA, USA). tGSH and GSSG concentrations of experimental samples were interpolated on calibration curves that were obtained from reactions containing either pure GSH or pure GSSG standards (0–16 μM tGSH or 0–8 μM GSSG). Results were given as GSSG over GSH ratio.
10,000× g for 15 min, at 4 °C. Cell lysates were deproteinized with 5% (w/v) metaphosphoric acid (cat. 239275, Sigma-Aldrich, Milan, Italy) and centrifuged at 4000× g for 5 min, as recommended by the supplier. In order to evaluate the abundance of GSSG, GSH in the samples was first derivatized with 2-vinylpyridine (cat. 132292, Sigma-Aldrich, Milan, Italy). Protein-free samples (50 µL) and the assay cocktail (150 µL) were loaded in a 96-well plate, with three replicates. Absorbance at 405 nm was followed for 30 min, with 5 min intervals, by using a Victor3 microplate reader (PerkinElmer, Waltham, MA, USA). tGSH and GSSG concentrations of experimental samples were interpolated on calibration curves that were obtained from reactions containing either pure GSH or pure GSSG standards (0–16 μM tGSH or 0–8 μM GSSG). Results were given as GSSG over GSH ratio.
10
Evaluating Cell Proliferation with DCA
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Cell proliferation/viability of LNCaP and DU-145 cells was assessed as described (39 (link)). 5x103 cells/well were plated in 96-well culture plates. A day after plating the cells, different concentrations of dichloroacetate (DCA; Sigma Aldrich) or vehicle were added. Cell proliferation was measured with the CellTiter 96®AQueous One Solution Cell Proliferation Assay Kit (MTS Assay, Promega, Madison, WI, USA) in 96-well plates, and luminescence was measured at 490 nm using Victor3 microplate reader (PerkinElmer, Waltham MA, USA). To assess cell confluence, 5x103 cells/well were seeded on 96-well plates and the cell confluence was measured every three hours by the IncuCyte FLR imaging microscopes (Essen Biosciences, Ann Arbor, MI, USA), as described (40 (link)). The cells were treated with the indicated DCA concentrations 21 h post-plating and were scanned for 72 h after adding the drug.
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