The mitochondrial transmembrane potential was analyzed using a JC‐1 Kit (Beyotime) and LDH release was measured via a LDH release kit (Beyotime) as we previously described23 .
Ldh release kit
Manufactured by Beyotime
Sourced in China
The LDH release kit is a laboratory equipment used to measure the activity of lactate dehydrogenase (LDH) released from cells. LDH is a common marker for cell damage or cell death. The kit provides the necessary reagents and protocols to quantify LDH levels in cell culture or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Jc 1 kit, by Beyotime (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Phospholipid assay kit, by Merck Group (1 mentions)
Fluorescein dutp, by Thermo Fisher Scientific (1 mentions)
One step tunel kit, by Beyotime (1 mentions)
Il 1β elisa kit, by Elabscience (1 mentions)
Elx800 universal microplate reader, by Agilent Technologies (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
15 protocols using ldh release kit
1
Mitochondrial Function and Cell Viability
2
Assessing Cell Viability and Membrane Integrity
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Cells were treated with TBM alone or combination with other drugs. After treatment, 0.5 mg/mL 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) was added and incubated for 4 h. The insoluble formazan product of cells was dissolved in dimethyl sulfoxide after three times washing with phosphate buffered saline (PBS). The optical density (OD) of each culture well was measured by spectrophotometry at 570 nm. The OD value of the control cells was taken as 100% viability. Cellular membrane integrity was assessed with the lactic dehydrogenase (LDH) Release kit (Beyotime), as described previously58 (link). Briefly, LDH activity was assayed by adding 100 μL potassium phosphate buffer within 23 mM pyruvate and 0.3 mg/mL β-NADH. Then the conversion of NADH to NAD+ was monitored at 340 nm with a spectrophotometer.
3
Photodynamic Therapy Enhances ATP and LDH Release
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4T1 cells (2 × 104) were incubated with COF samples (100 μg mL−1) for 24 h. The resulting samples were subjected to different treatments and irradiated under 660 nm (0.5 W cm−2, 5 min) and/or 808 nm (1.5 W cm−2, 5 min) lasers. The cell supernatants were collected after 6 h, and the extracellular release of ATP was measured using an ATP assay kit (S0026, Beyotime Biotechnology). The experimental LDH release was determined using an LDH release kit (C0016, Beyotime Biotechnology).
4
Quantifying Cytokine and Cytotoxicity in Ovarian Cancer
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ELISA assay was used to detect the level of IL-1β in human ovarian cancer cell lines by human IL-1β pre-coated ELISA kit (R&D, Shanghai, China). LDH release assay was performed using LDH release kit (Beyotime Biotechnology, Beijing, China) in accordance with the manufacture’s instruction. Data were collected using microplate reader (Thermo, Massachusetts, U.S.A.).
5
Bacterial Toxin Internalization Assay
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Two kinds of bacterial toxins including non‐hemolytic enterotoxin of B. cereus (Nhe) and α‐toxin of S. aureus (AT), were tested. Nhe (23 ng mL−1), AT (50 ng mL−1, Sigma‐Aldrich), and their corresponding neutralizing antibodies (anti‐NheB mAb 1E11, 2 µg mL−1 and anti‐α‐toxin mAb, MEDI4893*, 5 µg mL−1, Sigma‐Aldrich) were involved as well. Ribbit anti‐IgG antibody (2 µg mL−1, Beyotime) was used as a negative control. IEC‐6 cells, bacteria (B. cereus, S. aureus, and E. coli), and sole bacterial toxin in the presence or absence of its antagonist were simultaneously added. After incubation for 2 h, the numbers of internalized bacteria were counted as previous description above. Lastly, the damage of phospholipid resulting in increased levels of choline were detected by a Phospholipid Assay Kit (Sigma‐Aldrich) and lactate dehydrogenase (LDH) in the media from damaged cells were determined by a LDH Release Kit (Beyotime), according to their instructions.
6
LDH Release Cytotoxicity Assay
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The cytotoxicity under different treatments was assessed by using a lactate dehydrogenase (LDH) release kit (Beyotime, China). The studies were carried out in accordance with the instructions provided by the supplier.
7
TUNEL Assay for Apoptosis Detection
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A one-step TUNEL kit (Beyotime Institute of Biotechnology, Haimen, China) was used for TUNEL staining as previously described.28 (link) After the treatment, HepG2 cells were incubated with fluorescein-dUTP (Invitrogen; Thermo Fisher Scientific, Inc.) to stain apoptotic cell nuclei and DAPI (5mg/mL) to stain all cell nuclei at room temperature for 3 min. The slides were imaged under a confocal microscope at least 5 random separate fields. LDH release was measured using an LDH release kit (Beyotime, Beijing, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure cell viability as previously described.29
8
Evaluating Cellular Cytotoxicity and Inflammatory Response
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Cellular cytotoxicity was measured with an LDH release kit (#C0016, Beyotime, China) according to the manufacturer’s instructions. Cells were harvested at 24 h post-OGDR treatment, washed with cold PBS and centrifuged at low speed for 20 min. The supernatants were transferred to fresh tubes to measure the production of mature IL-1β using an IL-1β ELISA kit (Elabscience, USA). To assess CASP8 activity, the protein concentrations in BV2 cell lysates were measured with a BCA kit, and equal amounts of protein were mixed with the reaction reagent and added to 96-well plates for measurement with the CASP8 activity kit (BioVision, USA).
9
Cytotoxic Effect of BBD on Gastric Cancer Cells
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An LDH release kit (Beyotime, Shanghai, China) was used to analyze the cytotoxic effect of BBD on both MGC80-3 and AGS cells. The MGC80-3 and AGS cells were harvested with the indicated doses of BBD (0, 0.25, 0.5, 0.75, and 1 mg/mL) and incubated for 12 hours, 24 hours, or 48 hours in 12-well plates. Then, the LDH release assay kit was used to determine LDH activity in culture medium, in accordance with the manufacturer’s instructions. Specifically, the cells were first inoculated into 12-well plates (1.5 × 105 cells/mL). After they were cultured for 11 hours, 23 hours, and 47 hours in an incubator, LDH-releasing agent was added in each well of LDH-releasing groups and cultured continuously for 1 hour in an incubator. The supernatant (120 μL/well) was collected and added to a 96-well plate. Then, 60 μL/well of LDH-detection working fluid was added to each well, mixed thoroughly, and incubated for 30 minutes at room temperature (RT) in the dark. Finally, the optical density (OD) value was immediately measured at 408 nm using an ELISA reader.
10
Measuring Cell Death by LDH Release
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Cells were plated in a 24- or 96-well plate overnight. Then cells were transfected with VR1012 or VR1012-TauP301S. The transfection medium was replaced after 4 ~ 6 h and then were treated with compounds at indicated concentrations. After 24 h or 48 h of transfection, cell death was measured using lactate dehydrogenase (LDH) release kit (Beyotime) according to the manufacturer’s instructions. The percentage of cell death per well was calculated by comparing to that of the maximal cell death with LDH Release Reagent after deducting background signal in non-induced cells. The absorbance was measured using an ELx800 universal microplate reader (BIO-TEK).
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