The study was approved by Niigata University Ethical Committee(IRB No. 2620) and was carried out in accordance to the ethical principals of the Declaration of Helsinki. After obtaining informed consent, blood samples were obtained from healthy human volunteers. Peripheral blood mononuclear cells (PBMCs) were separated using Lymphocyte Separation Solution (Nacalai Tesque, Kyoto, Japan). For lymphokine-activated killer (LAK) induction, PBMCs were suspended at a concentration of 2 × 106 cells/ml in RPMI 1640 containing 5% FCS and IL-2 was added at a concentration 2000 U/ml. PBMCs were cultured in 25-cm2 tissue culture flasks for 4–6 days at 37 °C in a 5% CO2 atmosphere. T24 and HT1376 cells were treated with 2.5 μM of 9-ING-41 for 48 hours. Untreated cells were used as control. Cytotoxic assay was done using the CytoTox 96 Non-Radioactive Cytotoxic Assay (Promega, Madison, WI) according to the manufacturer’s instructions. Released lactate dehydrogenase (LDH) was measured at 490 nM using IMARK microplate reader (Bio-Rad Laboratories, Inc., Tokyo, Japan).
Cytotox 96 non radioactive cytotoxic assay
Manufactured by Promega
Sourced in United States
The CytoTox 96 Non-Radioactive Cytotoxic Assay is a colorimetric assay that quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. The assay provides a simple, accurate, and non-radioactive method to quantify cell cytotoxicity or cell-mediated cytolysis directly in culture supernatants.
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5 protocols using cytotox 96 non radioactive cytotoxic assay
1
LAK Induction and Cytotoxicity Assay
2
Cytotoxicity Assay for PBMC-Mediated Killing of RCC Cells
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Human peripheral blood mononuclear cells (PBMC) were separated from human blood obtained from healthy volunteers using Lymphocyte Separation Solution (Nacalai Tesque, Inc.). For activation, PBMCs were suspended at a concentration of 2×106 cells/ml in RPMI-1640 medium containing 10% FBS, and recombinant human interleukin 2 (IL-2; cat. no. 0617AFC12; Peprotech, Inc.) was added at a concentration of 2,000 IU/ml. PBMCs were cultured for 3 days at 37°C in a 5% CO2 atmosphere. CytoTox96® Non-radioactive Cytotoxic Assay (Promega Corporation) was used according to the manufacturer's instructions. The CytoTox96® colorimetric assay measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Briefly, RCC cells ACHN and Caki-1 and activated PBMCs were added to a round-bottom 96-well plate (Corning, NY) and mixed at the lymphocyte to cancer cell ratios between 1:2.5 and 1:80. Following 4-h incubation at 37°C, 50 µl of the supernatants were transferred to a fresh 96-well flat-bottom plate (Corning, Inc.), and the absorbance signal was measured at 490 nm using an iMark™ Microplate Reader. Experiments were performed in triplicate.
3
Assessing Macrophage Pyroptosis via LDH
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Pyroptotic cell death was measured by assessing LDH release in the cell culture supernatant of human and murine macrophages using a CytoTox 96 Non-radioactive Cytotoxic Assay (Promega) following the manufacturer’s recommended procedures.
4
Antigen-Specific Cytotoxicity Assay
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Splenocytes were collected 1 week after the final immunization and mixed with 2 × 106 naive splenocytes that had been previously treated with mitomycin C and cultured in the presence of P8 peptides (5 μg/ml) in a 24-well plate for 5 days at 37°C. The cells were washed twice with complete RPMI 1640 and then used as effector cells. Syngeneic naive splenocytes were prepared by adsorption of P8 peptides (5 μg/ml) and rPAL (5 µg/ml) for 3 days at 37°C, washed three times with complete RPMI 1640, and resuspended at a concentration of 5 × 106 cells per ml for use as target cells. The pulsed target (T) cells (1 × 104 cells/well) were added to a 96-well plate, and effector cells (E) were then added a E:T ratios of 50:1, 30:1, or 10:1. After incubation for 4 h, antigen-specific lysis was measured using the CytoTox 96® Non-Radioactive Cytotoxic Assay (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. The percent specific lysis was calculated as follows: % specific lysis = 100 × (experimental − spontaneous) / (maximal − spontaneous).
5
Assessing Adipocyte Membrane Permeability
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Plasma membrane permeability was determined by incubating adipocytes with 250 ng/ml PI and 5 μg/ml Hoechst 33342 for 10 min and then analyzing them under a microscope. Lysed (dead) cells, which released lactate dehydrogenase (LDH) were analyzed by CytoTox 96 Non-Radioactive Cytotoxic assay (Promega). Before analysis, adipocyte cells were cultured with DMEM medium without phenol red, supplemented with 5% FBS for 6 h, then 50 μl medium for assay by following the instruction from the kit. Dead cells were excluded by HMGB1 staining, thus d16 adipocytes were stained with Hmgb1 antibody and counterstained with DAPI.
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