Mouse TNF-α, IL-6, MIP-2, KC (Biolegend), Fibrinogen (Innovative Research), and HMGB1 (MyBiosource) specific enzyme linked immunosorbent assays (ELISA) were performed according to the manufacturer’s instructions. LDH cytotoxicity assay was performed using an LDH kit (Clontech) according to the manufacturer’s instructions.
Ldh kit
Manufactured by Takara Bio
Sourced in Japan
The LDH kit is a laboratory reagent used to measure the activity of the enzyme lactate dehydrogenase (LDH) in biological samples. LDH is an important marker enzyme found in various tissues, and its levels can provide insights into cellular damage or disease states. The kit provides the necessary components and protocols to quantify LDH levels through a colorimetric or spectrophotometric assay.
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Lab products found in correlation
Fibrinogen, by Innovative Research (1 mentions)
Hmgb1, by MyBioSource (1 mentions)
Mip 2, by BioLegend (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Gfp trap, by Proteintech (1 mentions)
0260 100 lc3 2g6, by Thermo Fisher Scientific (1 mentions)
35 mm imaging dishes, by Ibidi (1 mentions)
Multiskan go, by Thermo Fisher Scientific (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
9 protocols using ldh kit
1
Multiplex ELISA Cytotoxicity Assay
2
Huntingtin Aggregation and Cytotoxicity
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H4 neuroglioma cells were maintained, grown and transfected with HTT exon-1 fragments with a polyQ expansions of either 25 or 104 fused to GFP as in ref. 26 (link). Twenty-four hours after HTT25Q or HTT104Q transfection, cells in 35 mm imaging dishes (Ibidi), 60 mm or 100 mm dishes (Corning) were treated with vehicle (PBS) or MGO (0.5 mM) for 16 h. Total protein lysates were obtained as in ref. 13 (link). Htt was immunoprecipitated using GFP-trap (Chromotek) according to the manufacturer instructions. Widefield fluorescent microscope Zeiss Axiovert 200 M (Carl Zeiss MicroImaging) or point scanning confocal microscope Zeiss LSM 710 (Carl Zeiss MicroImaging) were used to visualize HTT inclusions. Immunobloting was performed according to standard procedures as in ref. 13 (link) using the following antibodies: anti GFP (NeuroMab, P42212, 1:3000); anti AGEs (Cosmobio, KAL-KH-001, 1:500); anti LC3 (Nano Tools, 0260-100/LC3-2G6, 1:2000) and anti β-actin (Ambion, AM4302, 1:5000). Cytotoxicity was measured by means of the LDH kit (Clontech), following manufacturer´s instructions. Cell viability was determined using the MTT assay according to standard procedures. Evaluation of HTT aggregation profiles by filter retardation assay was performed as in ref. 62 (link). Briefly, cellulose acetate membranes retain aggregates that are not soluble in SDS (1%) and that are larger than 0.22 μm.
3
LDH-Based Cytotoxicity Assay Protocol
4
LDH Activity Measurement in Serum
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The LDH activity in serum was detected by LDH kit (Clontech, Mountain View, CA, USA) following the instructions. Serum diluted in saline at a volume ratio of 1:10. Total 10 µL sample was added to the microplate. The substrate buffer and the oxidized coenzyme I solution were mixed at a volume ratio of 5:1, 60 µl of the mixed solution was added to each well. The sample was thoroughly mixed and stayed in 37 °C for 15 minutes. Add 50 µl of 2,4-dinitrophenylhydrazine solution and mix well with the sample. 37 degrees Celsius water bath for 15 minutes. 150 µl of stop reagent were added to samples and mixed well.
After standing at room temperature for 3 minutes, the wavelength was measured at 440 nm.
After standing at room temperature for 3 minutes, the wavelength was measured at 440 nm.
5
Quantifying Cell Death by LDH Assay
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Following the applied treatment, cell death was detected by lactate dehydrogenase (LDH) assay using a commercial available LDH kit (Takara, Tokyo, Japan). LDH release (×100 %) was calculated as follows: LDH in conditional medium/(LDH in conditional medium + LDH in cell lysates).
6
Quantifying Peptide-Induced Cell Cytotoxicity
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Enzyme activity was performed using the LDH kit (Takara- MK401) according to the manufacturer’s instructions. 1.0 × 104 cells/well were seeded in 96-well plate, peptide treatments at 5, 12.5, 25, 50 and 100 µM were added and incubation was carried out for 24 h. Next, cell culture supernatants were harvested and quantified for LDH levels at 490 nm through a MultiSkan Go (Thermo Fischer Scientific, Waltham, MA, USA). With the use of triton, 100% cell death was induced.
7
LDH-Based Epithelial Cell Death Assay
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Cell death was detected by LDH assay. LDH activity in the culture media of the control cells and the CSE-treated cells was determined using LDH kit (Cat. #MK401, Takara, Japan) according to the manufacturer's instructions. Light absorption was measured at wavelength of 490 nm using a SpectraMax M2 microplate reader (Molecular Devices, CA, USA). The relative LDH activity indicated the epithelial cell death and the data were plotted in a bar graph.
8
Quantifying Neuronal Membrane Injury
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When the cell membrane was injured after cell necrosis, intracellular enzyme LDH leaked into culture medium. The supernatants of neurons with different treatments were plated into a 96-well plate at 50 μL per well, with 3 duplicated wells with each treatment. LDH contents were measured according to the instructions of an LDH kit (Takara, Tokyo, Japan).
9
Quantifying Cardiomyocyte Necrosis via LDH Assay
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Cell necrosis induced by the H/R injury was evaluated by measuring the release of LDH, a biochemical indicator of cellular damage. Following experimental treatments, H9c2 cardiomyocytes were harvested and lysed, and the supernatants were then centrifuged for 15 min. LDH release in the supernatant was measured using a commercial LDH kit (Takara, Dalian, China) according to the manufacturer's protocols.
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