SHEPMYCN3 neuroblastoma cells (5 × 103 cells per well) were seeded in 96-well plates, transfected with control siRNA, ALYREF siRNA-1, or ALYREF siRNA-2 for 16–48 h with and without doxycycline. The mitochondrial membrane potential of cells was determined using a MitoProbe DilC1(5) Assay Kit (Life Technologies). The cells were incubated with DilC1(5) working solution (50 nM/well) for 30 min, and the fluorescence of DilC1(5) was measured at 630-nm excitation and 670-nm emission wavelengths using VICTOR Multilabel Counter (Perkin Elmer). Dead cells were determined by SYTOX Green staining (Life Technologies). The cells were incubated with SYTOX Green working solution (30 nM/well) for 30 min, and the fluorescence of SYTOX Green was measured at 490-nm excitation and 520-nm emission wavelengths using VICTOR Multilabel Counter (Perkin Elmer).
Victor multilabel counter
Manufactured by PerkinElmer
Sourced in United States
The Victor Multilabel Counter is a versatile laboratory instrument designed for high-throughput detection and measurement of various assays. It utilizes advanced detection technologies to provide accurate and reliable results for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Atplite, by PerkinElmer (5 mentions)
Viewplate, by PerkinElmer (3 mentions)
5 and 6 carboxy 2 7 dichlorodihydrofluorescein diacetate, by Thermo Fisher Scientific (2 mentions)
Carboxy h2dcfda, by Thermo Fisher Scientific (2 mentions)
Maxisorp nunc immuno plate, by Thermo Fisher Scientific (1 mentions)
Il 1β elisa kit, by R&D Systems (1 mentions)
Elisa, by RayBiotech (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Glycerol, by Euromedex (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Tween 80, by Merck Group (1 mentions)
Amino 2 deoxyglucose 2 nbdg, by Thermo Fisher Scientific (1 mentions)
Elisa kit, by Thermo Fisher Scientific (1 mentions)
Atplite 1 step kit, by PerkinElmer (1 mentions)
Alamarblue assay, by Thermo Fisher Scientific (1 mentions)
Intracellular ph calibration buffer kit, by Thermo Fisher Scientific (1 mentions)
P8074, by Merck Group (1 mentions)
Dy406, by R&D Systems (1 mentions)
32 protocols using victor multilabel counter
1
Mitochondrial Membrane Potential Assay in Neuroblastoma
2
Quantifying Intracellular ATP Levels
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A commercially available luciferase-luciferin system (ATPlite, Perkin Elmer) was used to measure the intracellular ATP levels, as previously described [25 (link), 26 , 73 ]. Briefly, SH-SY5Y cells were previously differentiated on 96-well ViewPlate (Perkin Elmer) and then exposed to the indicated treatments in DMEM medium. Primary rat cortical neurons were plated on 12 multiwell plates, then treated in Neurobasal medium according to assigned experimental groups, and finally lysed and transferred to a 96-well ViewPlate (Perkin Elmer) for the ATP assay. The intracellular ATP levels were analyzed with a luminescence counter (Victor Multilabel Counter, Perkin Elmer), normalized to the respective protein content, and expressed as percentages of the control value.
3
Quantification of IgG1 in B Cell Culture
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The concentration of IgG1 in B cell culture supernatants was assessed from 2 plates randomly chosen from total of 5 plates in ELISA assay. Maxisorp Nunc-Immuno Plates (Thermo Scientific, Waltham, MA) were coated with 50 μl per well of goat anti-human IgG1 (Fc) antibody (Thermo Scientific, Waltham, MA) at 4 μg/ml in PBS at 4 °C overnight. 50 μl per well of B cell culture supernatants was then added. Afterwards, 50 μl per well of HRP-conjugated goat anti-Human IgG1 (Southern Biotech Inc, Birmingham, AL) at 1:2,000 dilution was added as secondary antibody. Reaction was read at 450 nm on VICTOR Multilabel Counter (Wallac/Perkin Elmer, Waltham, MA). Purified human IgG1 whole molecule of known concentration (Pierce, Rockford, IL) with 3 fold dilutions starting at 10 μg/ml was used as standard curve.
4
Microbial Carbon Substrate Oxidation
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Biolog® GN2 (Biolog, Inc., Hayward, Calif, USA)
Water samples (0.15 mL) from each treatment stage were pipetted into each of the 96 wells (1 control and 95 different carbon substrates). During incubation microbes oxidize substrates in the plate wells and simultaneously reduce the colourless tetrazolium dye to a violet formazan.
Wells were scored for substrate oxidation after incubation for 14 days at 22 ± 2 ºC based on the fulfillment of 2 criteria: a visible violet coloration in the wells and an optical density (OD595nm) 30% or more above the control well value. Plates were read using a Victor Multilabel Counter (Perkin Elmer, Turku, Finland)
Water samples (0.15 mL) from each treatment stage were pipetted into each of the 96 wells (1 control and 95 different carbon substrates). During incubation microbes oxidize substrates in the plate wells and simultaneously reduce the colourless tetrazolium dye to a violet formazan.
Wells were scored for substrate oxidation after incubation for 14 days at 22 ± 2 ºC based on the fulfillment of 2 criteria: a visible violet coloration in the wells and an optical density (OD595nm) 30% or more above the control well value. Plates were read using a Victor Multilabel Counter (Perkin Elmer, Turku, Finland)
5
Quantifying Leptin and IL-1β Secretion
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Leptin secretion was measured with an enzyme-linked immunosorbent assay (ELISA) (Raybiotech) and detected with a biotinylated anti-leptin antibody, followed with streptavidin incubation. The IL-1β amount secreted by CD14+ monocytes was determined using the IL-1β ELISA kit (R&D Systems, Minneapolis, MN, USA). Samples were read on 450 nm with Victor multilabel counter (PerkinElmer, Waltham, MA, USA), and values were obtained by interpolating the results with a standard curve provided in both detection kits. Data obtained by an ELISA assay were normalized by cell number count before and after treatment.
6
Detecting Cell Surface Mfa1 Expression
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Cell surface expression of Mfa1 by the transconjugants was determined using an enzyme‐linked immunosorbent assay (ELISA) after adsorption of P. gingivalis strains onto Maxisorp plates (Nunc). Briefly, P. gingivalis cells were centrifuged at 3,000 g for 5 min and cell pellets were washed there times with PBS. Subsequently, 107 cells were incubated in each well for 2 hr at 4°C followed by washing with PBS to remove unbound bacteria. Bound cells were incubated with rabbit rMfa monoclonal antibodies (1:5,000 dilution) (Covance, Denver, PA) for 1 hr at 37°C. After washing with PBS, wells were reacted with horseradish peroxidase‐conjugated goat anti‐rabbit antibody (1:3,000 [ImmuneReagents Inc.]) for 1 hr at 25°C. Antigen–antibody binding was determined by adding 200 µl of 1× TMB ELISA substrate solution [3, 3′, 5, 5′‐tetramethylbenzidine] (Invitrogen) and the reaction was incubated for 15 min at 25°C. Reactions were stopped using 50 ul of stop solution (0.16 M H2SO4) and the endpoint was measured at 450 nm in a Victor Multilabel counter (Perkin Elmer).
7
Fluorescent Mycobacterium tuberculosis Strains
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Recombinant strains of Mtb H37Rv expressing an enhanced green fluorescent protein (GFP) or a red fluorescent protein DsRed [49 (link)] were cultured in Middlebrook 7H9 medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco), 0.2% glycerol (Euromedex), 0.05% Tween 80 (Sigma-Aldrich) and 50 μg/ml hygromycin (ThermoFisher Scientific) or 25 μg/ml kanamycin (Sigma-Aldrich) for H37Rv-GFP or H37Rv-DsRed, respectively. Cultures were maintained for 14 days until the exponential phase was reached. Before cell infection, bacilli were washed with Dulbecco’s Phosphate Buffered Saline (DPBS, free from MgCl2 and CaCl2, Gibco), resuspended in 10 mL RPMI-FBS and centrifuged at 1000 RPM for 2 min at room temperature to remove bacterial aggregates. Bacterial titer of the suspension was determined by measuring the optical density (OD600 nm) and GFP or DsRed fluorescence on a Victor Multilabel Counter (Perkin Elmer). The bacterial suspension was diluted at the required titre in RPMI 1640 supplemented with 10% FBS prior to infection. For in vivo studies, the non-fluorescent Mtb H37Rv strain were grown in Middlebrook 7H9 medium, as described previously [50 (link),51 (link)].
8
Quantifying Lipid Accumulation and Glucose Uptake in 3T3-L1 Cells
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Oil Red O (ORO) staining was performed on day 8. To measure the lipid accumulation in 3T3-L1 preadipocytes, cells were fixed with 10% formalin and stained with ORO. ORO dye was extracted by isopropanol to quantify relative TG accumulation [5 (link)]. The level of monocyte chemotactic protein-1 (MCP-1) in the supernatants were detected by using ELISA kit (Thermo Fisher Scientific Co., Rockford, IL, USA) according to the manufacture's protocol. The standard curve of MCP-1 was 16-1000 pg/mL. All standards and samples were assayed in triplicate. Cellular glucose uptake was quantified by the 2-[N-(7-notrobenz-2-oxa-1, 3-diazol-4-yl) Amino]-2-Deoxyglucose (2-NBDG, Invitrogen, Carlsbad, CA, USA) assay. 3T3-L1 cells were seeded in 96 well black plates and differentiated into mature adipocytes as reported previously [5 (link)]. On day 6, the cells treated with PPE. After 24 h, treatment medium was removed and added with 200 µL 2-NBDG solution for 30 min. Then, cells were washed twice with PBS, the cells were measured with a VICTOR™ Multilabel Counter (PerkinElmer, USA), set at an excitation wavelength of 465 nm and an emission wavelength of 540 nm [26 (link)].
9
ATP Synthesis Measurement in Cells
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ATP synthesis was evaluated using a commercially available luciferase-luciferin system (ATPlite, Perkin Elmer, Waltham, MA). The day before the experiment, cells were plated (5,000 cells/well) in 96 multiwell plates. The day after, cells were first washed with Tyrode’s solution containing (in mM): NaCl 137, KCl 2.7, MgCl2 1, CaCl2 1.8, NaH2PO4 0.2, NaHCO3 10, glucose 5.5 mM, pH 7.4 and then exposed to different glutamate concentrations (0.5 and 1 mM) in the same Tyrode’s solution for 1 h at 37 °C17. When ATP content was evaluated after H/R, glutamate and the specific pharmacological tools were added at the beginning of the reoxygenation phase and maintained for 1 h. After the incubation period, ATP levels were analyzed with a luminescence counter (Victor Multilabel Counter, Perkin Elmer) and normalized to the respective protein content17 (link),18 (link).
10
Intracellular ATP Quantification Assay
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The intracellular ATP content was evaluated by using a commercially available luciferase-luciferin system (ATPlite, Perkin Elmer) as previously described [15 (link),40 (link)]. Briefly, SH-SY5Y cells were plated on a 96-well “View Plate” (Perkin Elmer), subjected to specific treatments in culture medium and then analyzed for the ATP content according to the manufacturer’s protocol. Rat cortical neurons were plated on 12-well plates, and after the specific treatments, the neurons were appropriately lysed. Thereafter, 100 μL of the lysates were transferred to a 96-well View Plate to perform the ATP assay. The ATP levels were analyzed with a luminescence Victor Multilabel Counter plate reader (Perkin Elmer), normalized to the respective protein content, and expressed as percentages of the control value.
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