C1qA peptide (Biotin-GSKGEQGEPGAPGI, GenScript, Piscataway, NJ, USA), control peptide (Biotin-KAEQAEPAAPAI, GenScript), sDDR (2538-DR; R&D Systems), or BSA (Roche, Basel, Switzerland) were coated on an ELISA plate (Corning) at 8 μg/mL in PBS and incubated overnight at 4 °C. The plates were washed with 0.05% PBS with Tween-20 (PBST) twice and blocked with 1% BSA fraction V (Roche) in PBS at 25 °C for 1 h. Various concentrations of sDDR2 (0.1, 0.5, 2, or 8 μg/mL) or C1qA peptide (0.1, 0.5, 2, or 8 μg/mL) were added and incubated at RT for 2 h. After washing with 0.05% PBST, alkaline phosphatase (AP)-conjugated anti-6x-His Ab (3D5, Invitrogen, Waltham, MA, USA) for sDDR2 detection or AP-conjugated streptavidin (7105–04, Southern Biotech, Birmingham, AL, USA) for C1qA peptide or control peptide were incubated at RT for 1 h. Phosphatase substrate (S0942, Sigma-Aldrich) solution was added for color development, and the optical density of each well was measured at 405 nm.
S0942
Manufactured by Merck Group
Sourced in United States
The S0942 is a piece of lab equipment manufactured by Merck Group. It is designed for general laboratory use and its core function is to facilitate various experimental and analytical processes. The detailed specifications and intended applications of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Fast blue, by Merck Group (2 mentions)
Naphthol as mx, by Merck Group (2 mentions)
Bsa fraction 5, by Roche (1 mentions)
Elisa plate, by Corning (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
Phosphatase substrate, by Merck Group (1 mentions)
Ap conjugated streptavidin, by Southern Biotech (1 mentions)
Anti flag antibody, by Thermo Fisher Scientific (1 mentions)
M1000, by Tecan (1 mentions)
Infinite 200 pro microplate reader, by Tecan (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Enzchek phosphate assay kit, by Thermo Fisher Scientific (1 mentions)
Alamar blue solution, by Thermo Fisher Scientific (1 mentions)
Dal1100, by Thermo Fisher Scientific (1 mentions)
A8960, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
A5533, by Merck Group (1 mentions)
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
96 well elisa plate, by Thermo Fisher Scientific (1 mentions)
11 protocols using s0942
1
Enzyme-Linked Immunosorbent Assay for C1qA Peptide and sDDR2
2
Quantifying Membrane DOP Expression
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HEK293 cells (150 × 103) were transfected in suspension with 470 ng of DNA of the WT human DOP construct or one of the various mutants of the full-length DOP or the BRIL-DOP crystal constructs using X-tremeGENE HP 4:1 ratio in 24-well plates precoated with poly-l -lysine. Forty-eight hours after transfection, cells were fixed using a 3.7% formaldehyde/tris-buffered saline (TBS) solution. After 30 min of blocking with 1% BSA/TBS, the cells were incubated for 1 hour with a polyclonal anti-flag antibody (1:1000; Invitrogen, catalog no. 710662) followed by 1-hour incubation with anti-rabbit alkaline phosphatase (1:10,000; Sigma-Aldrich, catalog no. A3687). The level of expression of membrane DOP was detected using alkaline phosphatase substrate (Sigma-Aldrich, catalog no. S0942) in a solution containing 10% diethanolamine and 12.5 μM MgCl2 (pH 9.8). The reaction was stopped by the addition of 0.4 M NaOH before reading absorbance at 405 nm using a TECAN M1000 multimode reader. Results are expressed as means ± SD from n = 2 independent experiments, each performed in triplicate.
3
Anti-Human IgG Quantification Assay
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Reagents were added 100 µL/well in 96-well plates, coated overnight at 4°C with 0.1 µg/mL of hu14.18K322A hIgG1 (diluted in PBS buffer, pH 7.4). All wash steps were performed three times with 100 mM Tris 0.05%–Tween 20 (pH 7.4). Plates were blocked for 3 hours using PBS–5% milk, followed by a wash. Serum samples were diluted 1:250 in PBS–0.5% bovine serum albumin (BSA) and added in triplicate for overnight incubation at 4°C. Plates were washed; goat anti-mouse IgG alkaline phosphatase (Sigma ImmunoChemicals #A-4656) was added; and plates were incubated light protected for 2 hours at 20°C. Detection of anti-mouse IgG was emphasized due to the role of IgG in inducing memory and mediating effector functions via FcRs.15 (link) Plates were washed; 100 µL of p-nitrophenylphosphate (PNPP, alkaline phosphatase substrate, S-0942, Sigma) in diethanolamine buffer was added; and plates were incubated light protected for 1 hour at 20°C. The plates were read at 450 nm with a 570 nm reference filter. The standard curve reagent used for this assay was obtained by performing these measurements on serial dilutions of mouse anti-human IgG (BD Pharmingen #555784) in concentrations between 5 and 250 ng/mL in twofold increments.
4
ELISA Assay for Mouse Serum Antibodies
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Plates were coated with poly-l -lysine (Sigma-Aldrich) before addition of 2.5 µg of either DNA (D7290, Sigma-Aldrich), RNA (AM7120G, Thermo Fisher Scientific) or nRNP (SRC-1000, Immunovision). Plates were then blocked in ELISA blocking buffer (PBS and 1% BSA) for 2 h at room temperature. Mouse serum was diluted 1:40 with ELISA coating buffer (0.05 M sodium carbonate anhydrous/sodium hydrogen carbonate, pH 9.6), and incubated in the ELISA plates overnight at 4 °C. The plates were washed and goat anti-mouse IgG-AP antibodies (Alkaline Phosphatase, Southern Biotech) were added for 1 h at 37 °C. Phosphatase substrate (Sigma-Aldrich, S0942) was used as described by the manufacturer. The samples were read using the Infinite 200 PRO Tecan Microplate Reader (Tecan Group) at an absorbance of 405 nm and normalized to background absorbance at 605 nm.
5
Enzymatic Assay for HAD2 Phosphohydrolase
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The rate of para-nitrophenyl phosphate (pNPP; Sigma-Aldrich S0942) hydrolysis by HAD2 was determined by continuous measurement of absorbance at 405 nm. Assays were performed at 37°C in a 50-µl volume consisting of 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 10 mM pNPP, and 1.2 µM enzyme.
Hydrolysis of other phosphorylated substrates by HAD2 was measured using an EnzChek phosphate assay kit (Life Technologies). The reaction buffer was modified to contain 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 0.2 mM 2-amino-6-mercapto-7-methylpurine riboside (MESG), and 1 U/ml purine nucleoside phosphorylase (PNP). Reaction mixtures contained 5 mM substrate and 730 nM enzyme. The activity of catalytically inactive 6×His-HAD2D26A was measured for all substrates, and the data were used to normalize the activity found for the WT HAD2 enzyme. Activity was normalized to that obtained from catalytically inactive 6×His-HAD2D26A. All data represent means of results from ≥3 independent experiments performed with technical replicates.
Hydrolysis of other phosphorylated substrates by HAD2 was measured using an EnzChek phosphate assay kit (Life Technologies). The reaction buffer was modified to contain 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 0.2 mM 2-amino-6-mercapto-7-methylpurine riboside (MESG), and 1 U/ml purine nucleoside phosphorylase (PNP). Reaction mixtures contained 5 mM substrate and 730 nM enzyme. The activity of catalytically inactive 6×His-HAD2D26A was measured for all substrates, and the data were used to normalize the activity found for the WT HAD2 enzyme. Activity was normalized to that obtained from catalytically inactive 6×His-HAD2D26A. All data represent means of results from ≥3 independent experiments performed with technical replicates.
6
Osteoblast Characterization and Mineralization
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For alkaline phosphatase (ALP) staining, osteoblasts were fixed with 10% neutral formalin buffer and stained with the solution containing Fast Blue (Sigma, FBS25) and Naphthol AS-MX (Sigma, 855). Alternatively, osteoblasts were incubated with tenfold diluted Alamar Blue solution (Invitrogen, DAL1100) for cell proliferation. Subsequently, cells were washed and incubated with a solution containing 6.5 mM Na2CO3, 18.5 mM NaHCO3, 2 mM MgCl2, and phosphatase substrate (Sigma, S0942), and ALP activity was measured by luminometer (Biorad).
To assess extracellular matrix mineralization in mature osteoblasts, cells were washed twice with phosphate-buffered saline (PBS) and fixed in 70% EtOH for 15 min at room temperature. Fixed cells were washed twice with distilled water and then stained with a 2% alizarin red solution (Sigma, A5533) for 5 min. Cells were then washed three times with distilled water and examined for the presence of calcium deposits. Mineralization was quantified by the acetic acid extraction method52 (link).
To assess extracellular matrix mineralization in mature osteoblasts, cells were washed twice with phosphate-buffered saline (PBS) and fixed in 70% EtOH for 15 min at room temperature. Fixed cells were washed twice with distilled water and then stained with a 2% alizarin red solution (Sigma, A5533) for 5 min. Cells were then washed three times with distilled water and examined for the presence of calcium deposits. Mineralization was quantified by the acetic acid extraction method52 (link).
7
Osteoblast Differentiation and Mineralization
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For osteoblast differentiation, primary COBs were cultured in an osteogenic medium containing 0.1 mg/mL ascorbic acid (Sigma-Aldrich, A8960, Saint Louis, MO, USA), and 10 mM β-glycerophosphate (Sigma, G9422, USA), and incubated with Alamar blue solution (Invitrogen, DAL1100, Carlsbad, CA, USA; Dilution 1:10) for cell proliferation. Subsequently, cells were washed and incubated with a solution containing 6.5 mM Na2CO3, 18.5 mM NaHCO3, 2 mM MgCl2, and phosphatase substrate (Sigma-Aldrich, S0942 St. Louis, MO, USA), and alkaline phosphatase activity was measured by an iMark microplate absorbance reader (Bio-Rad, 1681130, Hercules, CA, USA). To assess extracellular matrix mineralization in mature osteoblasts, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Fixed cells were washed twice with distilled water and stained with a 2% Alizarin red solution (Sigma-Aldrich, A5533, St. Louis, MO, USA) for 10–20 min. Cells were then washed three times with distilled water and examined for the presence of calcium deposits.
8
Recombinant Yeast Cells Induce Antibodies
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Changes in humoral immune responses upon oral administration of recombinant yeast cells were analyzed by estimating the antibodies induced through indirect ELISAs using NUNC Maxisorp 96-well ELISA plates coated with 100 ng/well of E. coli-expressed recombinant scEDIII protein. The plates were washed three times with PBS + 0.05% Tween 20 and blocked with 1% BSA in PBS for 2 h at 37 °C. Following the washes, twofold serial dilutions were performed after adding 100 μL/well of sera from immunized mice (the starting dilution points were 1:25 for serum IgG and 1:2 for fecal sIgA), and the plates were incubated overnight at 4 °C. Alkaline phosphatase (AP)-conjugated secondary antibodies (anti-mouse IgG or IgA; Sigma-Aldrich) diluted 1:5000 in PBS containing 0.5% BSA were added and incubated for 2 h at 37 °C followed by a washing step. To detect the response, 100 μL/well of phosphatase substrate (S0942, Sigma-Aldrich) was added and incubated for 15 min at room temperature. The reaction was stopped with 2 M H2SO4 and the optical density was measured at 405 nm using a microplate reader (Multiskan™ GO Microplate Spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA).
9
Antibody Binding to Chikungunya Virus
10
Osteoblast Differentiation Analysis
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To assess osteoblast differentiation, alkaline phosphatase (ALP) activity was determined as described previously52 (link). In brief, differentiated osteoblasts were washed with phosphate-buffered saline (PBS) and incubated with a solution containing 6.5 mM Na2CO3, 18.5 mM NaHCO3, 2 mM MgCl2, and a phosphatase substrate (Sigma, S0942). ALP activity was measured using a spectrophotometer. ALP staining was performed using Fast Blue (Sigma, FBS25) and Naphthol AS-MX (Sigma, 855) after fixation in 10% neutral formalin buffer.63 (link)
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