Sodium hydroxide was purchased from Merck (Darmstadt, Germany); ethanol, hydrochloric acid, and dimethylsulphoxide were purchased from RCI Labscan Limited (Bangkok, Thailand); diethanolamine, albumin from bovine serum, CelLyticTM M, penicillin-streptomycin solution, and Bradford reagent were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA); magnesium chloride hexahydrate and 4-nitrophenol were purchased from Sigma-Aldrich Co. (Tokyo, Japan); 4-nitrophenyl phosphate disodium salt hexahydrate was purchased from Sigma-Aldrich, Co. (Dorset, UK); PrestoBlueTM cell viability reagent was purchased from Life Technologies Corporation (Eugene, OR, USA); Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline pH 7.4, fetal bovine serum (FBS), MEM non-essential amino acid solution, and 0.5% trypsin-EDTA solution were purchased from Life Technologies (Paisley, UK).
4 nitrophenyl phosphate disodium salt hexahydrate
Manufactured by Merck Group
Sourced in United States, Sweden
4-nitrophenyl phosphate disodium salt hexahydrate is a chemical compound commonly used as a substrate in various enzymatic assays, particularly in the detection and quantification of phosphatase enzymes. It serves as a colorimetric indicator, producing a yellow-colored product when cleaved by the enzyme, allowing for the measurement of enzyme activity.
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Lab products found in correlation
4 nitrophenol, by Merck Group (2 mentions)
Diethanolamine, by Merck Group (2 mentions)
Igg2c, by Southern Biotech (2 mentions)
Ap conjugated goat anti mouse adsorbed against human igg, by Southern Biotech (2 mentions)
Goat anti bovine igg, by Southern Biotech (2 mentions)
Maxisorp immunoplate, by Thermo Fisher Scientific (2 mentions)
Tween 20, by Merck Group (2 mentions)
Alp labeled rabbit anti mouse igg antibody, by Mabtech (2 mentions)
Elisa plate reader, by Tecan (2 mentions)
Azocasein, by Merck Group (2 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Albumin from bovine serum, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Hydrochloric acid (hcl), by RCI Labscan (1 mentions)
Magnesium chloride hexahydrate, by Merck Group (1 mentions)
Cellytic m, by Merck Group (1 mentions)
Ethanol, by RCI Labscan (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Igg2b, by Southern Biotech (1 mentions)
40 protocols using 4 nitrophenyl phosphate disodium salt hexahydrate
1
Reagent Purchasing and Cell Culture Protocol
2
Autoantibody Measurement by ELISA
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Anti-chromatin, anti-ssDNA, and anti-dsDNA IgM, IgG, IgG1, and IgG2a autoantibodies, were measured by ELISA, as previously described [6 (link)]. Briefly, 96 well flat bottom plates were coated with antigen and left overnight. Serum was diluted 1 in 50 and added in triplicate to the plates. Bound antibodies were detected using alkaline-phosphatase conjugated anti-IgM, -IgG, -IgG1, -IgG2a, IgG2b, IgG2c, or IgG3 secondary reagents (Southern Biotech; Birmingham, AL). Substrate (4-nitrophenyl phosphate disodium salt hexahydrate, Sigma-Aldrich, St Louis, MO, USA) was added, and the OD of each well was measured at a wavelength of 405 nm. Values were standardized from plate to plate by running known B6 and NZB controls.
3
ELISA Binding Assay of bRSV Antigens
4
Sporozoite Lysate Enzyme-Linked Immunosorbent Assay
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Sporozoite lysate was prepared by pelleting MAF purified sporozoites and flash freezing, before diluting in PBS and using to coat 96-well immunosorbent plates (NUNC MaxiSorp) overnight (1,500 sporozoites per well). Subsequently, liquid was removed and wells allowed to air dry before blocking with 1% BSA in PBS. Wells were subsequently incubated with mouse serum with starting dilutions of 1:50 or 1:100 in 0.01% Tween-PBS. Anti-mouse IgG secondary antibody conjugated to AP (Sigma-Aldrich) was added after five washes in 0.01% Tween-PBS. AP was quantified after five washes in 0.01% Tween-PBS using 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma-Aldrich) measured at 405 nm absorbance.
5
NETs-derived MPO Quantification by ELISA
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NETs remnant ELISA was conducted as previously described (14 (link)). Briefly, after coating 96-well microplates with a monoclonal mouse anti-nucleosome antibody (B6.SLE-1, 0.5 mg/mL) and blocking the additional binding sites, the plates were incubated with standards and serum for 2 h at room temperature. A rabbit anti-human MPO (1:500; DAKO, Carpinteria, CA, USA) was then added for 1 h at room temperature and an alkaline phosphatase-conjugated swine anti-rabbit antibody (1:500, DAKO) for another hour. Finally, a substrate for alkaline phosphatase (4-nitrophenyl phosphate disodium salt hexahydrate; Sigma-Aldrich, St Louis, MO, USA) was added, and the plates were read at 405 nm using a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). A standard curve was constructed for each plate, and all samples were interpolated from it.
6
RVFV Gn and Gc Ectodomain ELISA
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Recombinant RVFV Gn ectodomain (UniProt accession number P21401, residues 154–560) and Gc ectodomain (UniProt accession number P21401, residues 691–1120) were expressed in HEK293 cells as previously described [26 (link)]. This protein was used to perform ELISAs as previously described [26 (link)] with the exception of the following modifications. A positive reference serum made from a pool of high responding mice was included on each plate as a standard curve (titrated 1:2 and added in duplicate). The secondary antibody used (goat antimouse whole IgG-alkaline phosphatase, Sigma, St. Louis, MO, USA) was diluted 1:1000 in casein. Plates were developed by adding 100 µL/well of 4-nitrophenyl phosphate disodium salt Hexahydrate (Sigma) in diethanolamine buffer (ThermoScientific). Optical density (OD) was read at 405 nm using a BioTek ELx808 plate reader until the internal control (reference serum diluted 1:800) had reached an OD of 1. OD values of reference serum titrations were then fitted to a 4-parameter standard curve using Gen5 software (v3.09 BioTek). Test sera antibody units were calculated from their OD values using the estimated parameters from the standard curve.
7
Phosphatase Activity Assay
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Cells were incubated with 0.13% w/v 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma-Aldrich, Germany) dissolved in 0.1 M AP-buffer (50 mM glycine, 100 mM Tris-Base, 1 mM MgCl2 in dH2O) at pH 10.5 for 30 min at 37 °C and absorbance was measured at 405 nm wavelength.
8
Enzymatic Catalysis for Biochemical Synthesis
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All solutions were prepared using ultrapure water (MilliQ, Millipore, France). For use in synthesis by enzymatic catalysis, alkaline phosphatase isolated from bovine intestinal mucosa (ALP, lyophilized powder, ≥10 DEA units/mg solid), glycerol phosphate disodium salt hydrate (S, isomeric mixture, typically 50% β-isomer and 50% rac-α-isomer), calcium chloride (CaCl2, anhydrous, ≥99%), and sodium fluoride (NaF, BioXtra, ≥99%) were all sourced from Sigma-Aldrich (France). All solutions were prepared in 10 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, pH 7.4, made from Trizma® base (minimum 99.9% titration) and 1 N hydrochloric acid (HCl). For measurement of enzymatic activity, 4-nitrophenol (pnp, ReagentPlus® ≥ 99%) and 4-nitrophenyl phosphate disodium salt hexahydrate (pnpp, ≥99%) were sourced from Sigma-Aldrich. For pH adjustment, hydrochloric acid (HCl, 37%) from VWR and sodium hydroxide (NaOH, anhydrous pellets) from Carlo Erba Reagents were used.
9
Influenza Anti-antibody ELISA Protocol
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Blood was collected from all mice at two weeks post-immunization or four weeks post-immunization. Blood was allowed to clot at room temperature and then centrifuged at 180 × g for 15 min. Serum was collected and frozen at −80 °C until subsequent measurement of IgG, IgG1, and IgG2a anti-influenza antibodies by ELISA. Briefly, plates were coated overnight at 4°C with influenza virus A/PR/8/34 diluted in carbonate coating buffer (pH 9.6) at a 200 HA units/ml concentration for anti-influenza IgG, IgG1, and IgG2a. The wells were blocked with 0.1% BSA solution at 37 °C for one hour. Plates were washed three times with PBS containing 0.05% Tween 20 between each step. Diluted serum (1:50 for IgG and 1:5 for IgG1 or IgG2a) was added to the wells and incubated for 3 h at 37 °C. These dilutions were chosen based on preliminary assay optimization (data not shown). After incubation, AP-conjugated rat anti-mouse IgG, IgG1, or IgG2a were added (at 1:100 dilution for IgG and 1:10 for IgG1 and IgG2a), then plates were incubated overnight at 4 °C. Finally, phosphatase substrate (4-Nitrophenyl phosphate disodium salt hexahydrate, (Sigma) was added, and OD was assessed at 405 nm at 30 min (IgG) and 50 min (IgG1, IgG2a) using a Fluostar plate reader.
10
Quantifying Alkaline Phosphatase Activity
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Differentiation medium was removed, cell cultures washed with PBS, followed by addition of Tris‐EDTA buffer (0.1x). The plates were then stored at −80°C for at least 2 hours followed by thawing at room temperature. Para‐nitrophenylphosphate solution (2.5 mg 4‐nitrophenylphosphate disodium salt hexahydrate [Sigma] per 1 mL of buffer [0.1 M diethanolamine, 150 mM NaCl, 2 mM MgCl2]) was then added to each well. After a 30‐minute incubation at room temperature, absorbance was measured at 405 nm using the SpectraMAX Plus spectrophotometer (Molecular Devices). Raw values were then fit to a standard curve that was generated using reconstituted alkaline phosphatase enzyme (Roche, Indianapolis, IN, USA) to determine relative enzymatic activity within cultures.
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