For the ELISA analysis in bladder tissue, concentrations of TNF-α, IL-1β, and cAMP were measured by reacting with enzyme immunoassay kit (Abcam, Cambridge, UK) according to the manufacturer’s manual. For TNF-α and IL-1β measurement in the bladder tissue, standard or lysed sample was added to each well, and plates were incubated at room temperature for 2.5 hours. The prepared TNF-α and IL-1β biotin antibodies were added to each well and incubated at room temperature for 1 hour. Then, HRP-streptavidin solution was added and incubated at room temperature for 45 minutes. TMB one-step development solution was reacted at room temperature for 30 minutes in the dark on a shaker. Immediately, stop solution was added to each well and calculated at 450 nm wavelength with Multiskan Go Microplate Readers (Thermo Fisher Scientific). For cAMP calculation, prepared standard and diluted samples were added to wells. Then, prepared alkaline phosphatase-conjugate was added to wells. Subsequently, cAMP complete antibody was reacted at room temperature on a shaker for 2 hours (500 rpm). pNpp substrate solution was added to wells and incubated at room temperature for 1 hour. Then, stop solution was added and read at 405 nm wavelength with Multiskan Go Microplate Readers (Thermo Fisher Scientific).
Multiskan go microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States, Finland, China, United Kingdom, Japan
The Multiskan GO microplate reader is a compact and versatile instrument designed for a wide range of absorbance-based applications. It offers a wavelength range of 200 to 1000 nm and can read 6- to 384-well microplates. The Multiskan GO provides accurate and reliable measurements for various assays, including ELISA, cell-based, and biochemical analyses.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (8 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (3 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (3 mentions)
Uv 1800 spectrophotometer, by Shimadzu (3 mentions)
Cck 8 assay, by Dojindo Laboratories (2 mentions)
Celltiter 96 cell proliferation assay, by Promega (2 mentions)
Spd 20av uv vis detector, by Shimadzu (2 mentions)
Folin ciocalteau reagent, by Merck Group (2 mentions)
Cck 8, by Dojindo Laboratories (2 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Enzyme immunoassay kit, by Abcam (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Cell proliferation edu image kit, by Abbkine (1 mentions)
Cell counting kit 8 (cck8), by Apexbio (1 mentions)
Fitc conjugated antibodies, by BD (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Tnf α enzyme linked immunosorbent assay elisa kit, by Cusabio (1 mentions)
Cell counting kit 8 cck 8 kit, by Beyotime (1 mentions)
171 protocols using multiskan go microplate reader
1
ELISA Analysis of Inflammatory Markers in Bladder Tissue
2
Cell Viability Measurement by CCK-8 Assay
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Cell viability was measured by the cell counting kit-8 (CCK-8) assay (Dojindo, CK04, MD, USA). In briefly, cells were seeded in 96-well plates at 1×104 cells/well and stained with 10μl CCK-8/well at a certain point in time, after which optical density was detected at 450nm by Multiskan GO Microplate Reader (Thermo Scientific, Rockford, IL, USA).
3
MTT Assay for Cell Viability Assessment
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MTT assay was conducted as described by Mosmann (1983 (link)) with slight modifications. Confluent PBECs in 96-well plate were incubated with embelin prepared in the culture medium at concentrations ranging from 10 to 100 μg/mL, for 1 h at 37°C. After 1 h, embelin solution was discarded and the PBECs were incubated with 100 μL MTT solution (1 mg/mL) prepared in DMEM without Phenol Red for 4 h at 37°C. Untreated cells were used as control to represent total viable cells. The cells were also treated with 1% (v/v) methanol in culture medium as vehicle control. After 4 h, the MTT solution was removed and replaced with 100 μL of propan-2-ol to dissolve formazan crystals formed. Absorbance was measured at 560 nm and 690 nm using Multiskan Go Microplate Reader (Thermo Fisher Scientific, MA, USA). The experiment was conducted in triplicate, in three independent experiments. Percentage of cell viability was calculated using following equation:
4
Phosphorylated Tau Quantification in SH-SY5Y Cells
5
Cell Viability Assay with α-Arbutin NPs
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α-arbutin-loaded BMO-PUL NPs suspensions in DMEM (50 μL; 0.1–4 mg/mL) were cultured (37 °C) with confluent HaCaT cells for 24 h (seeding 5 × 103). The medium was then substituted with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (100 μL; 10% w/v) and incubated for another 3 h before being substituted by DMSO (100 μL). Initially, the MTT reagent was prepared by dissolving 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in a serum-free DMEM media (1 mg/mL). The plate was then analysed on a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA; measuring at 570 nm).
6
Cell Viability and Proliferation Assays
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The cell viability was determined by the Cell Counting Kit-8 (Apexbio, United States) according to the manufacturer’s instruction and read by the Multiskan GO microplate reader (ThermoFisher, United States ) at 450 nm. An amount of 800 cells per well was seeded in six-well plates and incubated for 14 days until colonies appeared. The colonies were fixed with methanol and stained with 0.5% crystal violet solution.
DNA synthesis was analyzed by using the EDU cell proliferation image kit (Abbkine, China). Briefly, 1 × 104 cervical cancer cells were cultured in 96-well plates in triplicate after transfection. cells were incubated with 50 μmol EdU for 2 h at 37°C, fixed with 4% paraformaldehyde for 30 min, then treated with 0.5% Triton X-100 for 10 min. The proliferation rate was determined under a fluorescence microscope.
DNA synthesis was analyzed by using the EDU cell proliferation image kit (Abbkine, China). Briefly, 1 × 104 cervical cancer cells were cultured in 96-well plates in triplicate after transfection. cells were incubated with 50 μmol EdU for 2 h at 37°C, fixed with 4% paraformaldehyde for 30 min, then treated with 0.5% Triton X-100 for 10 min. The proliferation rate was determined under a fluorescence microscope.
7
Colorimetric Assays for Mycoplasma Doubling Time
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Colorimetric assays used to measure M. florum doubling time were based on growth assays previously developed for spiroplasmas (Konai et al, 1996 ). Briefly, ATCC 1161 medium was inoculated with an exponential‐phase M. florum preculture to obtain an initial concentration of ~ 1 × 105 CFU/ml. The inoculated medium was then diluted using twofold serial dilutions to obtain a total of four dilutions (1:1, 1:2, 1:4, and 1:8). Each dilution was transferred in triplicate into a 96‐well microplate, and the plate was incubated with shaking at the desired temperature (30, 32, 34, 36 or 38°C) in a Multiskan GO microplate reader (Thermo Scientific). Bacterial growth was monitored by measuring the OD560 nm every 10 min for ~ 16 h. The metabolic activity of M. florum was previously shown to result in the acidification of the ATCC 1161 growth medium, causing changes in the absorbance of phenol red at 560 nm that correlate with the number CFUs (Matteau et al, 2015 ). To calculate doubling times, linear regressions (R2 > 0.999) were traced on the linear portion of the OD560 nm curves, and the amount of time separating each dilution curve was calculated according to the linear regression equations.
8
Characterization of Mesenchymal Stem Cells
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According to the criteria identified by Dominici et al. (2006) (link), cells were characterized by testing their plastic adherence, the immunophenotype and the multipotency.
For immunophenotyping, 2.5×105 cells were stained for 45 min with fluorescein isothiocyanate (FITC)-conjugated antibodies (Becton-Dickinson) against: HLA-DR, CD14, CD19, CD34, CD45, CD73, CD90, and CD105.
For differentiation assay, cells were induced toward osteocytes and adipocytes using STEMPRO® Osteogenesis and Adipogenesis Kits (GIBCO, Invitrogen), respectively (Orciani et al., 2013 (link)). Osteogenic differentiation was assessed by Alizarin Red staining after 10 days of induction; adipogenic differentiation was tested by Oil Red staining after 15 days of induction. Cells cultured in MSCGM alone were used as negative controls. For the quantification, Alizarin Red was detached by incubating with 10% cetylpyridinium chloride for 30 min at RT, then optical density was measured and quantified through a plate reader (Multiskan GO microplate reader, Thermo Fisher Scientific).
For immunophenotyping, 2.5×105 cells were stained for 45 min with fluorescein isothiocyanate (FITC)-conjugated antibodies (Becton-Dickinson) against: HLA-DR, CD14, CD19, CD34, CD45, CD73, CD90, and CD105.
For differentiation assay, cells were induced toward osteocytes and adipocytes using STEMPRO® Osteogenesis and Adipogenesis Kits (GIBCO, Invitrogen), respectively (Orciani et al., 2013 (link)). Osteogenic differentiation was assessed by Alizarin Red staining after 10 days of induction; adipogenic differentiation was tested by Oil Red staining after 15 days of induction. Cells cultured in MSCGM alone were used as negative controls. For the quantification, Alizarin Red was detached by incubating with 10% cetylpyridinium chloride for 30 min at RT, then optical density was measured and quantified through a plate reader (Multiskan GO microplate reader, Thermo Fisher Scientific).
9
Cell Proliferation Assay with CCK-8
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Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan) was used to assess cell proliferation. Ninety-six-well plates were seeded with 1 × 104 DLBCL cells per well for 24–72 h and then incubated with 10 μl of CCK-8 per well for 3 h. Measuring absorbance at 450 nm by the Multiskan GO Microplate Reader (Thermo Scientific, USA).
10
Immune Activation by Tumor-Derived DRibbles
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Lymphocytes derived from mice spleens were obtained as previously described 10 (link). Briefly, different concentrations (5, 10, 20, 40 μg/ml) of CT-26 cell-derived DRibbles were incubated with lymphocytes for 72 h. To investigate DCs loaded with tumor antigens, 20 μg/ml of CCSC-derived DRibbles (SD) and lysates (SL) and CT-26 cell-derived DRibbles (TD) and lysates (TL) were incubated with the splenic lymphocytes or CD8+ T cells (2 × 106 cells/well) for 72 h. Cell viability was assessed using a CCK-8 kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Cells treated with Concanavalin A (5 μg/ml) served as the positive control. Absorbance was measured at 450 nm using a Multiskan Go microplate reader (Thermo Fisher Scientific).
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