Wells were blocked (1% bovine serum albumen (BSA)), followed by 3 h incubation with zymogen (potent activator of the alternative complement pathway) in defibrinated plasma (serum), plus PolySia-NP or vehicle (10% sucrose) control. Complement deposition was visualized after 1 h incubation with human C3b antibody (Complement Tech-A114), then 20 min streptavidin-HRP (R&D Systems) incubation, and 20 min TMB substrate (R&D Systems) reaction. Results were read using a BioTek Synergy HTX plate spectrophotometer (Agilent) at 490 nm absorbance.
Synergy htx plate spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States
The Synergy HTX plate spectrophotometer is a compact and versatile instrument designed for a wide range of absorbance-based assays. It features a high-performance optical system and supports multiple detection modes, including UV-Vis and fluorescence. The Synergy HTX provides accurate and reproducible results across a variety of samples and applications.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin hrp, by R&D Systems (2 mentions)
Tmb substrate, by R&D Systems (2 mentions)
C3nab, by Merck Group (2 mentions)
Antibody sensitized sheep erythrocytes, by Complement Technology (1 mentions)
Elisa plates, by R&D Systems (1 mentions)
Nickel coated plates, by Thermo Fisher Scientific (1 mentions)
4 protocols using synergy htx plate spectrophotometer
1
Quantifying Complement Activation by PolySia-NP
2
Serum Complement Activation Assay
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Normal human serum (NHS; Complement Technology, Tyler, TX, USA) was treated with either nothing (untreated), sucrose vehicle control, PolySia-NP (0.3 mg/mL, 1 mg/mL), or C3 neutralizing antibody (C3nAb; Millipore, Burlington, MA, USA) and immediately used for CH50 assays. Normal human serum with cobra venom factor pre-activation (NHS-CVF) was included as a negative control. In brief, antibody-sensitized sheep erythrocytes (Complement Technology) were added to a dilution series of each serum sample in GVB++, incubated at 37 °C for 60 min, centrifuged to pellet remaining RBCs, transferred supernatants (containing heme from lysed RBCs) to 96-well plates, and read on a BioTek Synergy HTX plate spectrophotometer (Agilent, Lexington, MA, USA) at 560 nm absorbance. All data were normalized to the positive control at 100% (replace buffer with water or detergent) and negative control at 0% (no NHS). Six independent experiments were performed, and data were combined for presentation here.
3
Complement Activation Assay Using NHS
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Normal human serum (NHS, Complement Technology) was treated with either nothing (untreated), sucrose vehicle control, PolySia-NP (0.3 mg/mL, 1 mg/mL), or C3 neutralizing antibody (C3nAb, Millipore) and immediately used for AH50 assays. Normal human serum with C3 depletion (NHS-C3dpl, Complement Technology) was included as a negative control. In brief, rabbit erythrocytes (Complement Technology) were added to a dilution series of each serum sample in GVB plus MgEGTA, incubated at 37 °C for 30 min, centrifuged to pellet remaining RBCs, transferred supernatants (containing heme from lysed RBCs) to 96-well plates, and read on a BioTek Synergy HTX plate spectrophotometer at 560 nm absorbance. All data were normalized to positive control at 100% (replace buffer with water or detergent) and negative control at 0% (no NHS or add EDTA). Three independent experiments were performed, and data were combined for presentation here.
4
Measuring CFH Binding to Polysialic Acid Nanoparticles
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CFH-His (1 μg/mL; Abcam, Cambridge, UK, or Cloud Clone, Katy, TX, USA) was bound to nickel-coated plates (Thermo Fisher Scientific, Waltham, MA, USA) via overnight incubation at RT. CFH wild-type and Y402H polymorphism fragments (FH6,7/hIgG1Fc and FH6,7 Y402H/hIgG1Fc; 1 μg/mL each), generated as previously described [49 (link),50 (link)], were bound to ELISA plates (R&D Systems, Minneapolis, MN, USA) via overnight incubation at RT. Negative controls included PBS and recombinant human IgG-Fc (R&D Systems). Coated wells were washed and then PolySia-NPs (0.3–10 mg/mL) or blank NP negative control were incubated for 2 h, followed by washing, biotinylated PEG antibody (0.5 μg/mL; Abcam, Waltham, MA, USA) incubation for 1 h, washing, and streptavidin-HRP (R&D Systems) incubation for 20 min. PEG was then visualized using TMB substrate (R&D Systems) reaction, stopped (R&D Systems), and read at 490 nm absorbance using a BioTek Synergy HTX plate spectrophotometer. Four independent experiments were performed and data were combined for presentation here.
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