Restriction endonucleases, HindIII, BamHI, PstI, Phusion DNA polymerase, aLICator™ LIC Cloning and Expression System Kit 3, PageRulerTM Prestained Protein Ladder were purchased from Thermo Fisher Scientific, Vilnius, Lithuania, Pierce™ Coomassie Plus (Bradford, UK) Assay Reagent and HisPur™ Ni‐NTA spin column were purchased from Thermo Fisher Scientific, Rockford, IL. pET21a Vector was purchased from Novagen (Madison, USA). Nutrient medium were purchased Roth, Germany. pNP‐acyl esters, tributyrin, β‐d ‐glucose pentaacetate, β‐d ‐galactose pentaacetate, and uridine were purchased from Sigma‐Aldrich. ‘ZR Soil Microbe DNA MidiPrep™’ was purchased from Zymo Research, Freiburg, Germany. Nitrocefin was purchased from Oxoid, UK. 3′‐O‐benzoyl‐2′‐deoxyuridine were purchased from Carbosynth, UK. 3′‐O‐acetyl‐2′‐deoxyuridine, 3′‐O‐acetyl‐N4‐benzoyl‐2′‐deoxycytidine, 3′‐O‐levulinyl‐N4‐benzoyl‐2′‐deocytidine, and 5′‐O‐levulinyl‐N4‐benzoyl‐2′‐deocytidine were purchased from Jena Bioscience, Jena, Germany.
Assay reagent
Manufactured by Thermo Fisher Scientific
Sourced in Sweden
Assay reagents are chemical solutions used in laboratory settings to facilitate and support various analysis and testing procedures. These reagents provide the necessary components for specific assays, enabling accurate and reproducible measurements of target analytes. The core function of assay reagents is to enable the detection, quantification, or identification of substances within a sample. These reagents are an essential tool for researchers and scientists conducting a wide range of experiments and analyses in diverse fields such as biochemistry, molecular biology, and clinical diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocefin, by Thermo Fisher Scientific (1 mentions)
Uridine, by Merck Group (1 mentions)
Hispur ni nta spin column, by Thermo Fisher Scientific (1 mentions)
Pierce coomassie plus, by Thermo Fisher Scientific (1 mentions)
Tributyrin, by Merck Group (1 mentions)
β d glucose pentaacetate, by Merck Group (1 mentions)
β d galactose pentaacetate, by Merck Group (1 mentions)
Pagerulertm prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
3 o acetyl n4 benzoyl 2 deoxycytidine, by Jena Biosciences (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Hindiii, by Thermo Fisher Scientific (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
Nano glo luciferase kit, by Promega (1 mentions)
Ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Infinite 5 r f200, by Tecan (1 mentions)
5 protocols using assay reagent
1
Cloning and Protein Expression Protocol
2
Functional Validation of MPRA SNPs
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We designed and ordered oligonucleotides (IDT-DNA, Coralville, IA) containing the candidate MPRA-functional SNPs and 40bp of flanking genomic sequence (S5 Table ). The amplified sequence was inserted into nano-luciferase containing plasmid pNL3.2 (Promega, Madison, WI) using HindIII and XhoI (ThermoFisher, Waltham, MA) restriction sites. The sequence was confirmed by sequencing. Three independent preparations of reporter plasmids and β-gal expression plasmids were cotransfected in K562 cells or Meg-01 cells and luciferase assay was carried out after 48 hours using Nanoglo luciferase kit (Promega) and normalized to ß-gal expression measured using assay reagent (ThermoFisher).
3
Hippocampal TRPM7 Expression Assessment
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A small fraction of each hippocampus (35 mg) was homogenized in the radioimmunoprecipitation (RIPA) buffer lysis buffer (# MBS842826, MyBioSource, CA, USA) plus protease inhibitors. The supernatants were isolated (11,200× g/10 min/4 ℃) and protein levels were determined using an assay reagent (# 23225, ThermoFIsher, USA). Equal protein concentrations were separated using the SDS-PAGE. After successful transfer, the nitrocellulose membranes were incubated with the target antibodies (i.e TRPM7 and the loading control, β-actin) (# OST00031W, 240 kDa, 1:2000, TherromFisher Scientific, USA and # 24042, 45 kDa, 1:2000, Cell Signaling technology, MI, USA, respectively). Membranes were incubated with the horseradish peroxidase (HPR)-corresponding antibodies. The ECL Plus Western Blotting Substrate (# 32106, ThermoFisher Scientific, USA) was used as a developing agent and all images were captured and analyzed using the C-Di Git scanner and its available software.
4
Nanobiomaterial Cytotoxicity Evaluation
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Cytotoxicity of nanobiomaterials was evaluated by the Alamar blue (resazurin) assay as described (Keshavan et al., 2021 (link)). The cell viability experiments were performed by one of the participating laboratories, prior the “round robin” pre-validation experiments. The cells were trypsinized, counted, and resuspended in cell culture medium without phenol red and supplemented with 5% dextran-coated charcoal-stripped FCS (DCC–FCS), to a final concentration of 104cells/well (100 µl). Cells were seeded in 96-well plates and exposed to test materials or were maintained in DCC–FCS alone (negative control). The assay reagent (Thermo Scientific, Sweden) (10% [v/v] solution of AlamarBlue® reagent) was added to each well to monitor the cellular metabolic function. The samples were analyzed using a spectrophotometer (Tecan Infinite® F200).
5
Metabolic Activity Assessment of MWCNT Exposure
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Cells were seeded in 96-well plates in RPMI-1640 cell medium at a density of 10 6 cells/mL and exposed to MWCNTs at the indicated concentrations or were maintained in cell medium alone (negative control). The assay reagent (Thermo Scientific, Sweden) (10% [v/v] solution of AlamarBlue V R reagent) was added to each well to monitor the cellular metabolic function. The samples were analyzed using a spectrophotometer (Tecan Infinite V R F200). The experiment was performed with at least three biological and three technical replicates were applied for each concentration of MWCNTs.
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