A chemical library of 2839 compounds was purchased from MedChem Express (HY-L035, Monmouth Junction, NJ, USA). All chemicals were screened at 100 μM using the negative screening at first, whereby 100 μM isopropyl-β-d -1-thiogalactopyranoside was used to induce DP1 and MgM expression. Bacteria without isopropyl-β-d -1-thiogalactopyranoside (i.e., no protein induction) were used as a positive control, while bacteria that received only DMSO served as the negative control. Drugs that increased bacterial growth to a certain threshold were selected for further duplicate analyses. Subsequently, drugs that passed duplicate analysis in the negative assays were examined by positive assay, where 5 μM and 10 μM isopropyl-β-d -1-thiogalactopyranoside were used to induce DP1 and MgM expression, respectively. Compounds that passed the negative and positive assays were subjected to dose-response analyses and fluorescence-based assays. Finally, the negative assay tested the additive effects of drugs by employing equal concentrations of all possible combinations.
Hy l035
Manufactured by MedChemExpress
Sourced in United States
HY-L035 is a laboratory equipment product. It is a precision liquid handling device used for accurate and reproducible pipetting of small liquid volumes.
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Lab products found in correlation
5 protocols using hy l035
1
High-Throughput Chemical Library Screening for Protein Modulators
2
Screening for Steatosis Alleviators
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1600 drugs of a 2800-compound library (HY-L035, MedChem Express, NJ, USA) were screened for identification of novel potential therapeutic targets to alleviate steatosis. Cells were seeded in 96-well plates (20,000 cells/well) and 24 h later incubated with 0.5 Mm OA. 24 h later the drugs were introduced at a final concentration of 5 μM with 2.5% DMSO as control. After 24 h incubation, medium was removed and cells were washed and stained for DAPI and Nile Red. Triglyceride quantification was performed using a plate reader as describe above and in Supp. materials and methods .
3
High-Throughput Screening of Repurposed Drugs
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The chemical library was purchased from MedChem Express (HY-L035, Monmouth Junction, NJ, USA). At the time, the library contained 2839 repurposed drugs, noting that the number of chemicals changes with time. Each chemical was tested at a final concentration of 100 M. The final concentration of dimethyl sulfoxide was 2%. All manipulations and growths were conducted on a robotic system (EVO 75 Tecan, Männedorf, Switzerland).
For each growth test, two metrics were measured: maximal growth rate and final bacterial density. However, in practice, visual inspection was far superior in identifying individual hits due to spurious factors that may influence the aforementioned metrics, such as compound absorbance, solubility, etc.
The screening proceeded in three stages. Initially, we screened all compounds in the negative assay. Each plate had two controls: the positive control were bacteria without -d -1-thiogalactopyranoside, i.e., without channel induction. Blank DMSO addition served as a negative control. Subsequently, bacteria that experienced growth enhancement beyond an empirical threshold were reexamined in triplicate. Every compound that passed this assay was then examined in the positive assay in triplicate. Finally, compounds that passed both the positive and negative assays were subjected to a dose-response analysis in duplicate and the pH assay in quadruplicate.
For each growth test, two metrics were measured: maximal growth rate and final bacterial density. However, in practice, visual inspection was far superior in identifying individual hits due to spurious factors that may influence the aforementioned metrics, such as compound absorbance, solubility, etc.
The screening proceeded in three stages. Initially, we screened all compounds in the negative assay. Each plate had two controls: the positive control were bacteria without -
4
High-Throughput Screening of FDA-Approved Compounds for Cardioprotection
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The chemical screening was performed essentially as described previously (Du et al. 2022 (link)). Briefly, P3 NRVMs were isolated and cultured on 96-well plates at 15,000 cells per well. After 48 h, the culture medium was aspirated, and fresh culture medium (high-glucose DMEM containing 200 μΜ H2O2, 0.5% FBS, and 1% penicillin–streptomycin) with 2 μM small molecules was immediately applied. With regard to the preparation of 2 μM small-molecule plates, the chemical library arrayed in 384-well microplates consisted of 3,142 FDA-approved compounds (HY-L035, MedChemExpress, China); the compounds were transferred in a volume of 20 nL (10 mM) to 96-well plates using Echo 525 (Labcyte); and culture medium (100 μl DMEM containing 200 μM H2O2, 0.5% FBS, and 1% penicillin–streptomycin) was then distributed to each well using Multidrop Combi (Thermo Scientific), resulting in a final working concentration of 2 μM compounds. Both negative control (0.5% FBS) and positive control (10% FBS) were treated with 2 μM DMSO instead of compounds, while only the negative control was treated with H2O2. All of the NRVMs were then cultured for 24 h before LDH detection.
5
High-throughput Screening of Repurposed Drugs
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A library of 2839 repurposed drugs was purchased from MedChem Express (HY-L035, Monmouth Junction, NJ, USA). Note that the number of chemicals in the library changes with time. Each chemical was added at 100 µM concentration to the growth media with a total concentration of Dimethyl sulfoxide not exceeding 1%. All manipulations and growths were conducted on a Tecan EVO 75 robotic station (Männedorf, Switzerland).
At first, we screened all compounds in the negative assay using 96-well plates. Each plate had a positive and negative control. The positive control were bacteria in which channel expression was not induced, i.e., without β-d -1-thiogalactopyranoside. The negative control was bacteria to which DMSO was added without any chemicals.
Bacteria that exhibited growth enhancement above a certain empirical threshold were tested again in duplicate. Every compound that passed this test was then used in the positive assay in duplicate. Compounds that passed the positive and negative screens were then subjected to a dose–response analysis, as well as a fluorescence-based study.
At first, we screened all compounds in the negative assay using 96-well plates. Each plate had a positive and negative control. The positive control were bacteria in which channel expression was not induced, i.e., without β-
Bacteria that exhibited growth enhancement above a certain empirical threshold were tested again in duplicate. Every compound that passed this test was then used in the positive assay in duplicate. Compounds that passed the positive and negative screens were then subjected to a dose–response analysis, as well as a fluorescence-based study.
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