For quantitative ALP determination, cells were lysed in 0.2% (v/v) Triton X-100 in PBS for 30 min. ALP activity was measured by conversion of the p-nitrophenyl phosphate Liquid Substrate System (Sigma-Aldrich). The absorbance was measured at 405 nm and corrected at 655 nm on a multi-well plate reader (Bio-Rad, Hercules, CA, USA). Values were normalized to ALP activity in BMSCs cultured in BM.
Liquid substrate system
Manufactured by Merck Group
Sourced in United States
The Liquid Substrate System is a piece of laboratory equipment designed to facilitate the preparation and handling of liquid substrates. It provides a controlled environment for the storage, dispensing, and mixing of various liquid solutions used in laboratory experiments and assays. The system's core function is to enable the precise and consistent handling of liquid samples, helping to ensure the accuracy and reproducibility of experimental results.
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Lab products found in correlation
Multi well plate reader, by Bio-Rad (1 mentions)
Human myeloperoxidase duoset elisa, by R&D Systems (1 mentions)
Synergy 4, by Agilent Technologies (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Benchmark plus reader, by Bio-Rad (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Elisa plate, by Thermo Fisher Scientific (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Anti mouse igg, by Merck Group (1 mentions)
Novex, by Thermo Fisher Scientific (1 mentions)
Anti his antibody, by Thermo Fisher Scientific (1 mentions)
Prestained sds page standard, by Bio-Rad (1 mentions)
7 protocols using liquid substrate system
1
Quantitative Alkaline Phosphatase Assay
2
Quantifying Myeloperoxidase Levels: ELISA and Activity Assay
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MPO concentration was evaluated in plasma/urine samples using a commercial ELISA kit (Human Myeloperoxidase DuoSet ELISA - R&D Systems, Minneapolis, MN, USA) with standard range between 62.5–4,000.0 pg/mL. Plasma, but not urine, samples were diluted 1:100 in reagent diluent (1% BSA in PBS) and optical density was determined at 450 nm using in a microplate reader (Synergy 4–BioTek, Winooski, VT, USA). Intra-assay of 1.25% and 1.98% for urine and plasma analysis, respectively.
MPO activity was determined based in the method of Bradley et al.38 (link) by measuring tetramethylbenzidine (TMB) oxidation in the presence of hydrogen peroxide. Briefly, 30 µL of urine/plasma (plasma diluted 1:100 in PBS) were incubated with 20 µL of PBS and 100 µL of liquid substrate system (Sigma, St. Louis, MO, USA), composed by TMB and hydrogen peroxide, at 37 °C for 10 minutes protected from light. After incubation, the reaction was blocked with 100 µL sulfuric acid 2 N and optical density were determined at 450 nm. A standard curve (range 0.153–2500 mU/50 µL) was generated by incubation of horseradish peroxidase with the previous reagents. Intra-assay of 5.45% and 2.51% for urine and plasma analysis, respectively.
MPO activity was determined based in the method of Bradley et al.38 (link) by measuring tetramethylbenzidine (TMB) oxidation in the presence of hydrogen peroxide. Briefly, 30 µL of urine/plasma (plasma diluted 1:100 in PBS) were incubated with 20 µL of PBS and 100 µL of liquid substrate system (Sigma, St. Louis, MO, USA), composed by TMB and hydrogen peroxide, at 37 °C for 10 minutes protected from light. After incubation, the reaction was blocked with 100 µL sulfuric acid 2 N and optical density were determined at 450 nm. A standard curve (range 0.153–2500 mU/50 µL) was generated by incubation of horseradish peroxidase with the previous reagents. Intra-assay of 5.45% and 2.51% for urine and plasma analysis, respectively.
3
Aptamer-based ELISA for Trypanosoma Antigens
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ELISA plates were coated with 50 µL of 2.5 µg protein/well of TESA, trypomastigote or epimastigote extracts or with mice plasma samples diluted 1∶200 in PBS [19] (link). Coated plates were blocked with 1% bovine serum albumin (BSA) in PBS. After discarding the blocking buffer, biotinylated RNA aptamers were added to each well. After 1 hour incubation the plate was washed thrice with PBS to remove unbound aptamers. Streptavidin-alkaline phosphatase was added to the wells and bound conjugate detected using 4-Methyllumbelliferyl Phosphate (4-MUP) (Liquid Substrate System, Sigma) [19] (link). Fluorescence was measured at an excitation wavelength of 360 nm and emission wavelength of 440 nm, with a cutoff filter of 435 nm, using a Spectra Max, M5, (Molecular Devices) [19] (link).
4
HPV Pseudovirion Neutralization Assay
5
Western Blot Analysis of Nrf2 Signaling
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Protein expression level was analyzed using Western blot technique [37 (link)]. Cell lysates or nuclear fractions (in the case of phosphorylated protein Nrf2) were separated by 10% Tris-Glycine SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Following electrophoresis, separated proteins were transferred into a membrane and blocked with 5% skim milk. Primary antibodies against phospho-Nrf2 (pSer40), HO-1, Keap1, p21, p62, and peroxisome proliferator-activated receptors (PPARα, γ, and δ) (Sigma-Aldrich, St. Louis, MO, USA) were diluted 1:1000. Protein bands were visualized calorimetrically using the BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) Liquid substrate system (Sigma-Aldrich, St. Louis, MO, USA). Protein level was expressed as a percentage of control cells.
6
PPRV-Specific IgG ELISA Protocol
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Anti-PPRV IgG detection was performed as described previously (19 (link)). Concisely, 104 pfu per well of purified PPRV Nigeria 75/1 was used to coat ELISA plates (Maxisorp, Nunc) overnight at 4°C. Blockage and washes were performed with 10% FBS in PBS and 0.1% Tween in PBS, respectively. Sera from inoculated sheep were diluted in PBS + 1% FBS prior to addition to the plate. A secondary antibody donkey anti-sheep IgG conjugated with horseradish peroxidase (Serotec) diluted 1:6,666 in PBS + 0.5% FBS was used to detect PPRV-specific IgGs. A liquid substrate system (Sigma) of 3,3',5,5'-tetramethylbenzidine (TMB) was used as a developer agent, and reactions were stopped with 3 M sulfuric acid before reading. A FLUOstar Omega (BMG Labtech) ELISA plate reader was used to read at 450 nm the optical density (OD). The measurements were made in triplicate and considered valid with standard deviations below 10% of the average. Anti-PPRV IgG titer in serum was defined as the serum dilution necessary to achieve readings twice that of pre-immune serum from the same sheep and was calculated using a linear regression of serum dilutions vs. OD readings at 450 nm. Data are presented as the average (±SEM) IgG titer for each treatment group.
7
Immunoblotting of Recombinant SAG1 Protein
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to separate purified rSAG1 protein on a 12% polyacrylamide gel along-with protein molecular marker as a standard (pre-stained SDS-PAGE standards, BioRad, Hercules, CA, USA). After electrophoresis, resolved proteins were transferred from gel to nitrocellulose membrane (Novex (USA), Invitrogen) which was then immune-blotted with anti-SAG1 antibodies or serum of T. gondii-infected mouse. The dilution of serum was made 1:200 in 5% skimmed milk-TNT (comprising 0.05% Tween-20, 140 mM NaCl, 15 mM Tris-HCl) [51] . Immunoblot was detected using anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) or anti-HIS antibodies (Invitrogen, Waltham, MA) with alkaline phosphatase. The dilution of anti-antibodies was made 1:1000 in 5% skimmed milk-TNT. The catalytic activity of alkaline phosphatase was detected by using liquid substrate system (Sigma-Aldrich, St. Louis, MO, USA) i.e. 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium (BCIP/NBT).
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