Cell viability of irradiated cells was measured using MTT assay. Briefly, 1 × 104 cells were cultivated in a 96-well plate for 24 h and then exposed to irradiation. After 48 h post irradiation, MTT reagent (Sigma, Darmstadt, Germany) with a 5 mg/mL concentration was poured into each well and the cells were maintained at the 37 °C incubator for 4 h. Next, DMSO (dimethyl sulfoxide) compound was added to liquefy formazan crystals, and absorbencies were read at 570 nm using a microplate reader system (BioTek, Winooski, VT, USA).
Microplate reader system
Manufactured by Agilent Technologies
Sourced in United States
The Microplate reader system is a versatile laboratory instrument designed to analyze samples in a multi-well microplate format. It is capable of measuring various optical parameters, including absorbance, fluorescence, and luminescence, to quantify the presence or activity of specific analytes or biological processes within the samples.
Automatically generated - may contain errors
Lab products found in correlation
Mtt reagent, by Merck Group (1 mentions)
Duoset enzyme linked immunosorbent assay elisa kit, by R&D Systems (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Bio plex manager software, by Bio-Rad (1 mentions)
Vacutainer blood collection tube, by BD (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Roche (1 mentions)
7 protocols using microplate reader system
1
Cell Viability Assessment by MTT Assay
2
Multiplex Cytokine Profiling in Plasma
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Peripheral blood was collected in heparin-coated BD Vacutainer Blood Collection tubes (BD Biosciences). Blood samples were centrifuged within 12 hours of collection at 700g for 20 minutes at room temperature without brake. The top layer (plasma) was harvested and stored at −80°C until use. Plasma concentrations of 65 human cytokines/chemokines/growth factors were simultaneously measured using a magnetic bead-based multiplex kit (EPX650-10065–901; Invitrogen) according to the manufacturer’s instructions. The beads were read on a BioPlex 200 system (Bio-Rad). The standards at 4-fold serial dilutions were run on each plate in duplicate and used to calculate the concentrations of cytokines/chemokines/growth factors using the Bio-Plex Manager Software (Bio-Rad).
Plasma levels of soluble CD146 (sCD146), soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1), and intestinal fatty-acid binding protein (I-FABP) were quantified using the human CD146, ICAM-1, VCAM-1, and I-FABP DuoSet enzyme-linked immunosorbent assay (ELISA) kits (all from R&D Systems), respectively, according to manufacturer’s instructions. ELISA results were recorded using a microplate reader system (Bio-Tek).
Plasma levels of soluble CD146 (sCD146), soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1), and intestinal fatty-acid binding protein (I-FABP) were quantified using the human CD146, ICAM-1, VCAM-1, and I-FABP DuoSet enzyme-linked immunosorbent assay (ELISA) kits (all from R&D Systems), respectively, according to manufacturer’s instructions. ELISA results were recorded using a microplate reader system (Bio-Tek).
3
Quantifying Exosomes in Irradiated Cells
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In order to measure the number of exosomes in conditioned media (CM) of irradiated groups, the AhCE activity was examined using a commercial cholinesterase kit (Cat No. BXC080; Biorexfars, Karaj, Iran) according to manufacturer’s procedure. Briefly, after irradiation, MCF-7 cells were carefully washed with PBS and cultivated with FBS-free DMEM for 48 h. CMs were centrifuged at 15,000 g for 20 min at 4 °C. The reagent 1 (potassium hexacyanoferrate plus pyrophosphate) was mixed with CMs and incubated for 5 min at room temperature. Following the mixing with 2-butyrylthio-n,n,n-trimethylethanaminium iodide, the absorbance values were recorded at 405 nm at 3 different rest intervals by a microplate reader system (BioTek). AChE activity was calculated by applying the recommended formula: Activity (U/l) = 65,800 × ΔAbs/min.
4
Quantification of Inflammatory Markers
5
Exosome Quantification in Bronchoalveolar Lavage
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To quantity the number of exosomes in BAL samples, we performed the acetylcholine esterase assay (AChE activity) using a commercial cholinesterase kit (Cat no. BXC080, Iran) according to the company’s guidelines. In brief, 100 μl of BAL samples were mixed with 500 μl R1 buffer (pyrophosphate + potassium hexacyanoferrate) and kept for 5 min at room temperature. Then, 20 μl R2 buffer (S-butyryl thiocholine iodide) was added, and optical density was obtained at 405 nm by three different intervals using a microplate reader system (BioTek). AhCE activity was measured by the following formula: Activity (U/l) = 65,800 × ΔAbs/min.
6
ELISA Quantification of Cytokines
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ELISA was performed on the BALF supernatant to determine the concentration of cytokines (IL-6, -8, -10, -17A, and-22, TNF-α, TGF-β). The levels of IL-17A, -22, and -10 in the cell culture supernatant were also assessed according to the kit’s instructions. The detection kits of mouse IL-6 (MEIMIAN14206), IL-8 (MEIMIAN4103), TNF-α (MEIMIAN920), IL-17A (MEIMIAN9401), IL-22 (MEIMIAN12178), TGF-β (MEIMIAN21034), and IL-10 (MEIMIAN4502) were bought from Jiangsu Enzyme Industry Co., Ltd. (Nanjing, China). The optical density (OD) value at 450 nm wavelength was determined using a BioTek Microplate Reader System (BioTek, Winooski, VT, USA).
7
Melatonin Cytotoxicity Evaluation in Breast Cancer Cell Lines
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MTT assay was applied to evaluate IC50 of Melatonin. First, the cells (10 4 cells/well) were plated in 96well culture plates. After incubation of the cells with different concentrations of melatonin, the media was replaced with fresh media containing 2 mg/ml MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) (Roche Molecular Biochemicals, Indianapolis, IN) solution and incubated for an additional four h at 37 °C. Then, the solution was replaced with a 200µl DMSO solution. Finally, the absorbance value was measured at 570 nm using a Micro-plate reader system (BioTek, USA). IC50 was determined for each agent by calculating the slope and intercept of different concentrations. The experiment was performed in triplicate and repeated three times. After determining the IC50 values for melatonin, all experiments were performed in 6 groups, including control groups of MCF-7 and MDA-MB-231 cell lines and melatonin treated groups(0.625mM) absence or presence of EGF (100 ng/mL) for 48h.
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