Classical complement pathway activation was assessed using EshA in Gelatin Veronal Buffer supplemented with Ca2+ and Mg2+ (GVB++; 10 mM Barbital, 145 mM NaCl, 0.1% Gelatin, 0.5 mM MgCl2, 0.15 mM CaCl2, pH 7.3 ± 0.15; Boston BioProducts, Ashland, MA, USA) (Morgan, 2000 ). In brief, approximately 5 × 106 EshA were incubated with serially diluted NHS (20% to 2.5%) and different titrations (0–50 μM) of cisplatin or pyridostatin in GVB++ at 37ºC for 10 min. For negative controls, 5 mM EDTA was added to the buffer to inhibit complement activation. For the alternative pathway, rabbit erythrocytes (Erabb) and Gelatin Veronal Buffer supplemented with Mg2+ & EGTA (GVB-Mg-EGTA; 10 mM Barbital, 145 mM NaCl, 0.1% Gelatin, 0.5 mM MgCl2, 10 mM EGTA, pH 7.4 ± 0.2; Boston BioProducts,) were used (Morgan, 2000 ). All the other conditions were the same as that in the classical pathway. After incubation, the erythocytes were centrifuged after the incubation, and the optical density (OD) at 414 nm (OD414) was measured in each 80 μl aliquots of supernatants containing released hemoglobin and used to calculate the percentage of hemolysis as follows: Hemolysis rate (%) = [(A - B)/(C - B)] × 100%, where A is the OD414 of tested samples, B is the OD414 of negative controls with EDTA, and C is the OD414 of maximum hemolysis induced by H2O.
Gelatin
Manufactured by Boston BioProducts
Sourced in United States
Gelatin is a translucent, brittle, colorless, flavorless solid substance, derived from the partial hydrolysis of collagen obtained from the skin, bones, and connective tissues of animals. It is commonly used as a gelling agent in food, pharmaceutical, and photographic industries.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin r phycoerythrin pe, by Agilent Technologies (2 mentions)
Red fluorescent neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Ique screener plus, by Intellicyt (1 mentions)
Lyophilized guinea pig complement, by Cedarlane (1 mentions)
Barbital, by Boston BioProducts (1 mentions)
Neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Freshly reconstituted guinea pig complement, by Cedarlane (1 mentions)
6 protocols using gelatin
1
Complement Pathway Activation Assay
2
Quantifying Antibody-Mediated Complement Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described recently (32 (link)), biotinylated antigen gp120 MN was coupled to red fluorescent neutravidin beads (Thermo Fisher Scientific). Immune complexes were formed with plasma samples for 30 minutes at 37°C at 3 dilutions (1:100, 1:500, 1:2500) in order to calculate the AUC. Lyophilized guinea pig complement was resuspended according to manufacturer’s instructions (Cedarlane), and 2 μL per well was added in veronal buffer with 0.1% gelatin (Boston BioProducts). After 20 minutes of incubation at 37°C, immune complexes were washed and C3 was detected with a fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (catalog 855385, MpBio). Complement-coated beads were acquired on a BD LSR II with a high-throughput sampler, and C3 deposition was reported by gating on single beads and C3+ events of 2 independent runs. AUC was calculated in Prism.
3
Quantifying SARS-CoV-2 RBD Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described in (59 (link)), the selected ACE2-Fc variants at varied concentrations (0.02–5 μg/mL) were incubated with multiplex assay microspheres coated with SARS-CoV-2 RBD or S-6P for 2hr at RT. Lyophilized guinea pig complement was resuspended according to manufacturer’s instructions (Cedarlane), and 2 μL per well was added in veronal buffer with 0.1% gelatin (Boston BioProducts). After washing, the mixtures of ACE2-Fc/microspheres were incubated with guinea pig complement serum at RT with shaking for 1 h. Samples were washed, sonicated, and incubated with goat anti-guinea pig C3 antibody conjugated with biotin (Immunology Consultants Laboratory) at RT for 1 h followed by incubation with streptavidin R-Phycoerythrin (PE, Agilent Technologie) at RT for 30min. After a final wash and sonication, samples were resuspended in Luminex sheath fluid and complement deposition was determined on a MAGPIX (Luminex Corp) instrument to define the median fluorescence intensity (MFI) of PE from two independent replicates. Assays performed without ACE2-Fc and without complement serum were used as negative controls.
4
Multiplex Assay for ACE2-Fc Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described in (27 (link)), the selected ACE2-Fc variants at varied concentrations (0.02 to 5 μg/ml) were incubated with multiplex assay microspheres coated with SARS-CoV-2 RBDwt, RBDB1.1.529, or S-6P for 2 hours at RT. Lyophilized guinea pig complement was resuspended according to the manufacturer’s instructions (Cedarlane), and 2 μl per well was added in veronal buffer with 0.1% gelatin (Boston BioProducts). After washing, the mixtures of ACE2-Fc/microspheres were incubated with guinea pig complement serum at RT with shaking for 1 hour. Samples were washed, sonicated, and incubated with goat anti–guinea pig C3 Ab conjugated with biotin (Immunology Consultants Laboratory) at RT for 1 hour followed by incubation with streptavidin R–phycoerythrin (PE; Agilent Technologie) at RT for 30 min. After a final wash and sonication, samples were resuspended in Luminex sheath fluid, and complement deposition was determined on a MAGPIX (Luminex Corp.) instrument to define the median fluorescence intensity of PE from two independent replicates. Assays performed without ACE2-Fc and without complement serum were used as negative controls.
5
Antibody-mediated Complement Deposition Assay
6
Quantifying Anti-ZIKV NS1 Antibody Complement Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant ZIKV NS1 protein (Native Antigen) was biotinylated and coupled to red fluorescent Neutravidin beads (Life Technologies) and then incubated with serial dilutions of anti-NS1 mAbs for 2 h at 37 °C to allow for binding. Freshly reconstituted guinea pig complement (Cedarlane Labs) was diluted 1 : 10 in veronal buffer containing 0.1% gelatin (Boston Bioproducts), which then was incubated with the antibody-bead complexes for 20 min at 37 °C. After washing in 15 mM EDTA in PBS, the complexes were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-guinea pig C3b antibody (1 : 100; MP Biomedicals 0855385) for 15 min at room temperature. Complement deposition was detected using an IntelliCyt iQue Screener Plus or a 3 L Stratedigm S1300EXI flow cytometer, and the median fluorescent intensity of FITC was measured for all beads using FlowJo software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!