The cytotoxicity of JGF on BHK-21 and Calu-3 cells was determined using Cytotoxicity Detection KitPLUS (LDH; Merck). BHK-21 and Calu-3 cells were seeded in 96-well plates (1×104 cells/well). After incubation overnight at 37°C, the cells were replaced into growth medium containing various concentrations of JGF (20, 40, and 80 μg/ml), and incubated at 37°C for 24 h. The untreated cells were as the low control so they spontaneously release LDH in normal condition. Cells that were treated with lysis buffer (5 μl) for 15 min were the high control and were used to determine the maximum release of LDH in the cells. To determine the LDH activity, 100 μl of Reaction mixture (freshly prepared by mixing Catalyst and Dye solution) was added to each well and incubated for 15 min at room temperature. Multimode microplate readers (TECAN SPARK) were used to measure the absorbance of the samples at a wavelength of 490 nm. To determine the percentage of cytotoxicity, the average absorbance values for three samples and controls were calculated. These values were substituted into the equation:
Cytotoxicity detection kitplus
Manufactured by Merck Group
Sourced in France, United States
The Cytotoxicity Detection KitPLUS is a laboratory product designed to assess the cytotoxic effects of various substances on cell cultures. The kit provides the necessary reagents and protocols to quantify the release of lactate dehydrogenase (LDH), a stable cytoplasmic enzyme, which is a reliable indicator of cell membrane damage and cell death.
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15 protocols using cytotoxicity detection kitplus
1
Cytotoxicity Evaluation of JGF on Cell Lines
2
Evaluating Cell Cytotoxicity and Viability
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To evaluate cell cytotoxicity by the extracellular lactate dehydrogenase (LDH) activity, Cytotoxicity Detection KitPLUS was used according to the manufacturer's protocol (4744934001; Merck). For MT‐ES‐MLs, 5 × 103 cells were seeded into each well of 384‐well plates, and then compounds in DMSO at a concentration of 100 nM were applied. Three hours later, 100 ng/mL IFN‐γ was added for the stimulation. After culturing for 21 hours, the supernatants were sampled. Detections were performed by using POWERSCAN4. For MT‐iPS‐MLs and fibroblasts, 1 × 104 MT‐iPS‐MLs and 1.5 × 103 NNS#1 fibroblasts were seeded into each well of 96‐well plates. After culturing for 24 hours, the supernatants were sampled. Detections were performed by using 2104 EnVision Multilabel Plate Readers (PerkinElmer). In every case, total cell lysates (100% cell death) from the same number of cells were used as the positive control.
To evaluate cell viability by the intracellular nicotinamide adenine dinucleotide (NADH) activity, 5 × 104 MT‐iPS‐MLs or 5 × 103 NNS#1 fibroblasts were seeded into each well of 96‐well plates. After culturing for 24 hours, Cell Counting Kit‐8 (CK04; Dojindo) was used according to the manufacturer's protocol.
To evaluate cell viability by the intracellular nicotinamide adenine dinucleotide (NADH) activity, 5 × 104 MT‐iPS‐MLs or 5 × 103 NNS#1 fibroblasts were seeded into each well of 96‐well plates. After culturing for 24 hours, Cell Counting Kit‐8 (CK04; Dojindo) was used according to the manufacturer's protocol.
3
Cytotoxicity Evaluation Using LDH and Cell Viability Assays
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To evaluate cell cytotoxicity by the extracellular lactate dehydrogenase (LDH) activity, Cytotoxicity Detection Kit PLUS was used according to the manufacturer's protocol (4744934001; Merck). For MT-ES-MLs, 5ൈ10 3 cells were seeded into each well of 384-well plates, and then compounds in DMSO at a concentration of 100 nM were applied. Three hours later, 100 ng/mL IFN-γ was added for the stimulation.
After culturing for 21 hours, the supernatants were sampled. Detections were performed by using POWERSCAN ○ R 4. For MT-iPS-MLs and fibroblasts, 1ൈ10 4 MT-iPS-MLs and 1.5ൈ10 3 NNS#1 fibroblasts were seeded into each well of 96-well plates. After culturing for 24 hours, the supernatants were sampled.
Detections were performed by using 2104 EnVision Multilabel Plate Readers (PerkinElmer). In every case, total cell lysates (100% cell death) from the same number of cells were used as the positive control.
To evaluate cell viability by the intracellular nicotinamide adenine dinucleotide (NADH) activity, 5ൈ10 4 MT-iPS-MLs or 5ൈ10 3 NNS#1 fibroblasts were seeded into each well of 96-well plates. After culturing for 24 hours, Cell Counting Kit-8 (CK04; Dojindo) was used according to the manufacturer's protocol.
After culturing for 21 hours, the supernatants were sampled. Detections were performed by using POWERSCAN ○ R 4. For MT-iPS-MLs and fibroblasts, 1ൈ10 4 MT-iPS-MLs and 1.5ൈ10 3 NNS#1 fibroblasts were seeded into each well of 96-well plates. After culturing for 24 hours, the supernatants were sampled.
Detections were performed by using 2104 EnVision Multilabel Plate Readers (PerkinElmer). In every case, total cell lysates (100% cell death) from the same number of cells were used as the positive control.
To evaluate cell viability by the intracellular nicotinamide adenine dinucleotide (NADH) activity, 5ൈ10 4 MT-iPS-MLs or 5ൈ10 3 NNS#1 fibroblasts were seeded into each well of 96-well plates. After culturing for 24 hours, Cell Counting Kit-8 (CK04; Dojindo) was used according to the manufacturer's protocol.
4
Evaluating Cell Viability Assays
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Cell viability in NCI-H292 cells were evaluated by use of the MTT assay and in HBECs were evaluated by the lactate dehydrogenase (LDH) activity using Cytotoxicity Detection KitPLUS (Sigma-Aldrich) as previously described [26 (link)]. Cell viability was calculated as the percentage of viable cells among vehicle-treated cells.
5
Membrane Damage Assessment by LDH Leakage
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Membrane damage induced by the different DHA lipid carriers was assessed by lactate dehydrogenase (LDH) leakage into the culture medium. The presence of LDH in the extracellular medium reflects a defect of membrane permeability which may result from necrosis and/or cell lysis. The activity of LDH in the medium was determined using the Cytotoxicity Detection Kit Plus (Sigma Aldrich, Saint Quentin Fallavier, France). The absorbances at 490 nm and 600 nm were read using a BioTek Epoch 2 microplate reader spectrophotometer system. Following the manufacturer’s instructions, the cytotoxicity of the tested molecules was calculated relative to a low control (spontaneous LDH release) and a high control (maximum LDH release) according to the equation: cytotoxicity (%) = [(Aassay − Alow control)/(Ahigh control − Alow control)] × 100, where A = A490 − A600. Each molecule was tested in triplicate wells, and three independent experiments were performed.
6
Analyzing Effector T Cell Cytotoxicity
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To examine the cytotoxicity of effector T cells, we i.p. injected the Nile tilapia individuals with 1 × 107 293T cells on day 1 and day 3. The animals were treated with indicated inhibitors on day 1, 3, and 5, and spleen T cells were sorted on day 6 for the assay. 2 × 106 T cells were incubated with 2 × 105 293T cells or NIH3T3 cells at 28 °C for 8 h. Cytotoxicity was assessed as LDH release, using the Cytotoxicity Detection Kit Plus (Catalog #4 744 926 001, Sigma) according to the manufacturer's instructions.
7
Cytotoxicity and Immune Response Profiling
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The quantified levels of IFN-γ, TNF-α, Granzyme B secreted by co-cultured activated CD8+ T cells were detected using ELISA kits, including Human TNF-α ELISA Kit, IFN-γ ELISA Kit (MultiSciences, China) and Human Granzyme B ELISA Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. The supernatants were collected and detected by 450 nm Optical densities using microplate reader (Bio-Tek). For the surface PD-L1, ~ 80% confluency BC cells were stained by FAM-labeled PD-L1 antibody (BioLegend, USA). After washing by PBS three times, FAM-positive cells were detected by flow cytometry. The release of lactate dehydrogenase (LDH) in the supernatants was detected on cell lysis using the Cytotoxicity Detection Kit PLUS (Sigma-Aldrich) according to the manufacturer’s protocol.
8
Neutrophil Degranulation and Death
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Resting neutrophils (Donor g–i) were inoculated with S. aureus (LS1, multiplicity-of-infection (MOI) 1:5, bacteria:neutrophils), 37 °C, 0–120 min. Resting neutrophils without S. aureus inoculation and with cytochalasin B/ionomycin (CytB/I) and Triton-X 100 stimulation served as activation and a cell death control, respectively. Degranulated MPO (Dg-MPO) and cell death were monitored longitudinally in supernatants using ELISA (ICL LAB) and lactate dehydrogenase (LDH) release using the Cytotoxicity Detection KitPLUS (Sigma), see Extended experimental procedures for details.
9
Evaluating T Cell-Mediated Tumor Cytotoxicity
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For cell cytotoxicity, activated T cells were co-cultured with tumor cells in a 5:1 ratio in the presence of different concentrations of F7AK3 for indicated times. Afterwards, T cell cytotoxicity was assessed by quantification of lactate dehydrogenase (LDH) amounts in the supernatants using the Cytotoxicity Detection KitPLUS (Sigma-Aldrich) according to manufacturer’s instructions.
For measurements of T cell activation and cytokine expression, PBMCs from human donors were cocultured with tumor cells in an Effector:Target (E:T) ratio of 3:1 in the presence or absence of F7AK3 (1 µg/mL) for 72 hours. T cell activation were assessed by incubating cells with directly conjugated antibodies to CD4 (Biolegend, 980812), CD8 (BD, 557746), CD69 (BD, 560738), CD25 (Biolegend, 302603), PD1 (BD, 558694). For cytokine expression, cocultured cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin and Golgiplug for 4 hours and antibodies against interferon γ (IFN-γ) (BD, 563287), tumor necrosis factor α (TNF-α) (Biolegend, 502944), Granzyme B (Invitrogen, 1893847), IL-2 (Biolegend, 500322) were used for intracellular staining. Data were analyzed by flow cytometry with a FACS LSR II or FACS Verse (BD).
For measurements of T cell activation and cytokine expression, PBMCs from human donors were cocultured with tumor cells in an Effector:Target (E:T) ratio of 3:1 in the presence or absence of F7AK3 (1 µg/mL) for 72 hours. T cell activation were assessed by incubating cells with directly conjugated antibodies to CD4 (Biolegend, 980812), CD8 (BD, 557746), CD69 (BD, 560738), CD25 (Biolegend, 302603), PD1 (BD, 558694). For cytokine expression, cocultured cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin and Golgiplug for 4 hours and antibodies against interferon γ (IFN-γ) (BD, 563287), tumor necrosis factor α (TNF-α) (Biolegend, 502944), Granzyme B (Invitrogen, 1893847), IL-2 (Biolegend, 500322) were used for intracellular staining. Data were analyzed by flow cytometry with a FACS LSR II or FACS Verse (BD).
10
LDH Release Measurement via Cytotoxicity Kit
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The LDH release into the media was measured using the Cytotoxicity Detection KitPLUS (Sigma, St. Louis, MO). Cells were subjected to OGD/R or normoxia, as explained above, then media were collected after the end of the reperfusion. The media were centrifuged at 250 g for 10 min and then processed to measure the LDH release, following the manufacturer's instructions.
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