To determine the effects of MRGD and MRGDE on cell injury and oxidative stress induced by LPS-ENR, the cells were inoculated in 6-well plates at 3 × 105 cells/mL and divided into 4 groups (BLANK, LPS-ENR, MRGD, and MRGDE groups). The drugs (MRGD, MRGDE) were added to the MRGD and MRGDE groups for 12 h, respectively. Then LPS (30 μg/mL) and ENR (125 μg/mL) were mixed and added to the LPS-ENR, MRGD, and MRGDE groups for 12 h, respectively. The cell viability was detected by the MTT assay and a lactate dehydrogenase (LDH ) kit (BC0685, Solarbio, Beijing, China). The oxidative stress of cells was estimated by detecting the malondialdehyde (MDA ) level (A003-4-1, Nanjing Jiancheng, Nanjing, China) and superoxide dismutase (SOD ) (A001-3-2, Nanjing Jiancheng, Nanjing, China) and catalase (CAT ) activities (A007-1-1, Nanjing Jiancheng, Nanjing, China) following the detection kits protocol of Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Ldh kit
Manufactured by Solarbio
Sourced in China
The LDH kit is a laboratory assay used to quantitatively measure the activity of the enzyme lactate dehydrogenase (LDH) in biological samples. LDH is an important indicator of various disease conditions and cellular damage.
Automatically generated - may contain errors
Lab products found in correlation
A001 3 2, by Nanjing Jiancheng (1 mentions)
A003 4 1, by Nanjing Jiancheng (1 mentions)
A007 1 1, by Nanjing Jiancheng (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Ab133595, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab287839, by Abcam (1 mentions)
Ab203516, by Abcam (1 mentions)
Ab178012, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Ripa protein lysate, by Thermo Fisher Scientific (1 mentions)
Ab182733, by Abcam (1 mentions)
Bca protein concentration kit, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Cck 8 reagent, by Beyotime (1 mentions)
Triton x 100 solution, by Merck Group (1 mentions)
8 protocols using ldh kit
1
Mitigating LPS-Induced Oxidative Stress
2
Evaluating 3DVGs Anticoagulant Properties
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The anticoagulant ability of 3DVGs in vitro was determined by platelet and recalcified whole blood adhesion assay. Fresh rabbit whole blood was maintained in the anticoagulant tubes containing 3.8% sodium citrate. After the addition of calcium chloride (CaCl2) solution (100 mM), 3DGs were incubated with recalcified whole blood for 30 min. The 3DGs with clotted blood were rinsed with PBS and weighed. The PRP was obtained by centrifuging the rabbit whole blood at 200g for 15 min. 3DGs were incubated with PRP at 37°C for 1 hour. After gently rinsing the 3DGs with PBS, the amount of platelets adhered to 3DGs was determined by an LDH kit (BC0685, Solarbio, Beijing, China) according to the instructions. Then, the morphology of 3DGs with platelet and recalcified whole blood adhesion was examined by SEM.
Fresh rat whole blood was drawn into EDTA anticoagulation tubes. We added DTβ4 (10 μg/ml), mixed well, took 10 μl, and diluted to 1 ml with PBS. We also added 2 μl of CD62P (BioLegend, B311813) and CD41 (BioLegend, B282877) antibodies, incubated for 2 hours in the dark, and then detected using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, USA).
Fresh rat whole blood was drawn into EDTA anticoagulation tubes. We added DTβ4 (10 μg/ml), mixed well, took 10 μl, and diluted to 1 ml with PBS. We also added 2 μl of CD62P (BioLegend, B311813) and CD41 (BioLegend, B282877) antibodies, incubated for 2 hours in the dark, and then detected using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, USA).
3
Cell Lysis and LDH Assay Protocol
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After the H/R treatment, the culture medium was removed and pre-cold PBS was
utilized to wash cells. Extract regent was added according to the number of
cells (10 4 (link) ) and the volume of extract (mL) at a ratio of 500:1. Then, cells were
disrupted on ice, centrifuged at 8000g for 10 min at 4°C. LDH levels of cell
supernatant were immediately detected with LDH kit (Solarbio, Beijing, China)
under a spectrophotometer.
utilized to wash cells. Extract regent was added according to the number of
cells (10 4 (link) ) and the volume of extract (mL) at a ratio of 500:1. Then, cells were
disrupted on ice, centrifuged at 8000g for 10 min at 4°C. LDH levels of cell
supernatant were immediately detected with LDH kit (Solarbio, Beijing, China)
under a spectrophotometer.
4
Cytotoxicity Assessment via LDH Release
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As a marker of cytotoxicity, the LDH level in cell supernatant was detected by a lactate dehydrogenase kit (LDH kit; Solarbio) on day 1, 3, 5 and 7. After collecting and centrifuging the cell supernatant, the activity of LDH was tested, and then calculated with the standard curve and relative optical density (OD) values.
5
Ox-LDL Cytotoxicity Evaluation in HUVECs
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HUVECs were cultured in the 96-well plate and grew to confluence of about 80%. After treatment with the ox-LDL at various concentrations (0–120 μg/ml) for 24 h, LDH activity in the culture medium was detected based on the manufacturer’s protocol of the LDH kit (catalog no.: BC0685; Solarbio).
6
Modulation of Cardiomyocyte Survival Signaling
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AC16 human cardiomyocytes, sourced from the BeNa Culture Collection (Beijing, China). MiR-449b-5p mimics, its inhibitor, negative control (NC), si-BCL2L13, si-NC, and BCL2L13-overexpressing plasmid (pcDNA3.1-BCL2L13) and vector control were purchased from Ribo Biological Co., Ltd. (Guangzhou, China). CCK-8 reagent (Beyotime, Shanghai, China), LDH Kit (Beijing Solarbio Science & Technology Co., Ltd.) and FITC-Annexin V/PI kit (BD Biosciences, San Jose, CA, USA) were purchased from corresponding companies. MDA (ab287797), ROS (ab287839), SOD (ab178012), specific primary antibodies against BCL2L13 (ab203516), Bcl-2 (ab196495), Bax (ab182733), PI3K (ab133595), p-PI3K (ab182651), AKT (ab8805), p-AKT (ab8933) and GAPDH (ab9485) were provide by Abcam (Cambridge, UK). Horseradish peroxidase-conjugated secondary antibodies (SC-2054 SC-2054) was purchased from Santa Cruz Inc., (Santa Cruz, CA, USA). RIPA protein lysate was from Thermo Fisher Scientific, Inc.) and BCA protein concentration kit was from Beyotime. Other inorganic reagents were purchased from Sigma-Aldrich.
7
Quantifying Cardiomyocyte Lysis by LDH Assay
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Following various treatments, we lysed AC16 cardiomyocytes using 1% Triton X-100 solution (Sigma-Aldrich) for 20 min and then centrifuged at 400 x g for 5 min at 4 °C to discard cellular debris. We measured the LDH levels in the supernatant using a commercial LDH Kit (Beijing Solarbio Science & Technology Co., Ltd., cat. no. BC0685), following the provided instructions.
8
Quantifying Neuronal Damage via LDH Release
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Previous findings have shown that the activity of the LDH released from damaged cells is proportional to the number of damaged neurons (McCord and Fridovich, 1969 ; Wang et al., 2015 (link)). After exposure to the drugs, 50 μl of the medium was removed, and the amount of LDH released from the cells was determined using a LDH kit (Solarbio Life Sciences, China) according to the manufacturer’s instructions. The absorbance of the samples was read spectrophotometrically at 450 nm. The results are expressed as the percentage of LDH released relative to that released from the control cells.
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