Allylamine was obtained from Sigma-Aldrich (Saint Louis, MO, USA). LDH assay was provided by Sigma-Aldrich. BSA were provided by Amresco (Solon, OH, USA). The 316L stainless steels were purchased from Baowu (Shanghai, China). Glutaraldehyde aqueous solution (30%) and ethanol were obtained from Keshi (Chengdu, China). Phosphate-buffered saline tablet was provided by Sigma-Aldrich. Other reagents were local products of analytical grade.
Ldh assay
Manufactured by Merck Group
Sourced in France, United States
The LDH assay is a laboratory test used to measure the activity of the enzyme lactate dehydrogenase (LDH) in a sample. LDH is an enzyme found in various cells throughout the body, and its levels can indicate the presence of certain medical conditions.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay, by Bio-Rad (2 mentions)
Allylamine, by Merck Group (1 mentions)
Phosphate buffered saline tablet, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Elisa quantitation set, by Fortis Life Sciences (1 mentions)
Model 680, by Bio-Rad (1 mentions)
Synergy h1 plate reader, by Agilent Technologies (1 mentions)
Alamarblue assay, by Thermo Fisher Scientific (1 mentions)
Centrifugal filter unit, by Merck Group (1 mentions)
Quercetin, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Mtt assay, by Promega (1 mentions)
Elisa reader, by Tecan (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
16 protocols using ldh assay
1
Cytotoxicity Evaluation of Allylamine
2
Cell Viability Assessment via TUNEL and LDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was tested via two methods; The DeadEndTM Fluorometric TUNEL system (Promega) and LDH assay (Sigma). Cell death was assessed through the TUNEL assay, according to the manufacturer’s protocol. Nuclei were stained with Hoechst (Life technologies). LDH activity in culture medium, representing relative cell viability and membrane integrity, was measured by spectrophotometer using the LDH assay kit, following the manufacturer’s instructions.
3
Cytotoxicity Assay for AKG Screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytotoxicity assay was conducted to estimate the non-cytotoxic concentrations of AKG against SiHa, CaSki, SW948, SW620, and CCD 841 CoTr cells. Briefly, cells plated in 96-well plates at a density of 3 × 105 cells/mL (SiHa, Caski, SW948), 5 × 105 cells/mL (SW620), and 2.0 × 105 cells/mL (CoTr) were treated with AKG at concentrations of 6.25, 12.5, 25, 50, 75, and 100 mM for 48 h. Untreated control cells (0 mM) were cultivated with appropriate cell culture medium with 2% of FBS. The cytotoxicity of AKG was estimated by means of the LDH assay (Sigma-Aldrich) [24 (link)]. The assay was performed according to the manufacturer’s instruction. The optical density was determined with the use of an EL800 Universal Plate Reader (Bio-Tek Instruments, Inc., USA). The level of LDH release was expressed as a percent of untreated cells. The highest concentrations of AKG that were non-cytotoxic to all the cells, i.e., 12.5, 25, and 50 mM, were chosen for further experiments (Supplementary Figure S1 ).
4
Quantifying Vascular Leakage and Cell Death
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vascular leakage into BAL fluid was assessed using a mouse serum albumin ELISA Quantitation Set (Bethyl Laboratories, Inc., Montgomery, TX). To measure cell death, lactate dehydrogenase (LDH) activity was quantified using an LDH assay (Sigma-Aldrich). This assay was performed in a 96-well plate for 30 min according to the manufacturer's instructions and analyzed using the MicroTek plate reader (Bio-Rad, Hercules, CA).
5
Assessing C60(OH)24 Cytotoxicity in A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was determined using MTT assay. Briefly, A549 cells were grown on 96-well plates at a density of 5 × 104 for 24 hours. After treatment with increasing doses of C60(OH)24 (10–200 μM) for 48 hours and 72 hours, cells were incubated with MTT solution (0.5 mg/mL, 1 × PBS) for 2 hours at 37°C. The reaction was then terminated by the addition of 100 μL dimethyl sulfoxide. The formazan crystals resulting from mitochondrial enzymatic activity on MTT substrate were solubilized with 200 μL of dimethyl sulfoxide, and absorbance at 570 nm was measured using a microplate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA). For assessment of cell membrane integrity, lactate dehydrogenase (LDH) release in exposure medium was measured with LDH assay following the manufacturer’s instructions (Sigma-Aldrich). The results are given relative to the untreated control.
6
Quantifying Membrane Integrity via LDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LDH activity was quantified in cell culture medium at different time points (30 min and 1, 2, 4, 6, and 24 h) after treatment. It is an accurate way to determine plasma membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. For that purpose, LDH Assay (Sigma, #TOX7, St Quentin Fallavier, France) was performed according to the manufacturer's protocol. Note that to perform a reliable quantification, fetal calf serum added to cell culture medium has to be heat‐inactivated (30 min at 56 °C), as it contains LDH residual activity.[47 ] Briefly, 45 µL of supernatant were mixed with 90 µL of LDH mix (Substrate Solution: Dye Solution: Cofactor Preparation) in 96‐well plates, incubated protected from light at room temperature for 20 min before absorbance was read at 490 nm on a Synergy H1 plate reader (Biotek, Winooski, VT, USA).
7
Cell Viability and Cytotoxicity Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was tested with two methods: alamarBlue assay (Invitrogen) and LDH assay (Sigma-Aldrich). Cell death was assessed through alamarBlue assay according to the manufacturer's protocol. LDH activity in culture medium representing relative cell viability and membrane integrity was measured using the LDH assay kit spectrophotometrically following the manufacturer's instructions.
8
Characterization of Lung Inflammation and Injury
9
Solubilization of Quercetin with Bile Salts and Phospholipids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quercetin (anhydrous), sodium oleate (OA), bile salts (sodium taurocholate (NaTC) dried to constant weight over calcium oxide and sodium glycodeoxcholate (NaGDOC)), pyrene (99.9%), ascorbic acid and dimethyl suphoxide (DMSO) were from Sigma-Aldrich (Gillingham, UK). DMSO, Quercetin and OA were stored under argon. Phosphatidylcholine (PC) made from egg lecithin (grade 1) and lysophosphatidylcholine (lysoPC) made from egg lecithin were from Lipid Products (South Nutfield, UK). Dulbecco’s phosphate buffered saline (DPBS), Ca2+ and Mg2+ free, (10×) were from Gibco, Life Technologies Corp (Paisley, UK). The LDH assay was from Sigma-Aldrich. Other chemicals were of analytical or HPLC grade. Centrifugal filter units (30 K molecular weight cut-off, size 2 and 15 mL) were supplied by Merck Millipore (Darmstadt, Germany).
10
Cytotoxicity Assessment of Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Various cell types cultured on 96-well plates were subjected to HEPES-mediated transfection. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; cells subjected to the transfection were incubated for 24 h and then centrifuged at 300 × g for 5 min. The supernatant media were discarded, and the attached cells were analyzed using a commercially available MTT assay (Promega, Madison, WI, USA). Cell toxicity was assessed using the lactate dehydrogenase (LDH) assay. To this aim, the cells subjected to the transfection were incubated for 48 h and then centrifuged at 300 × g for 5 min. The supernatant media were then collected and analyzed using a commercially available LDH assay (Sigma). The MTT and LDH signals were measured on an ELISA reader (Tecan, San Jose, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!