ADCD was adapted from methods described previously (76 (link)). Briefly, SARS-CoV-2 S-expressing expi293F cells were incubated with 10-fold diluted, heat-inactivated (56 °C for 30 min) plasma samples for 30 min at 37 °C. Cells were washed twice and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS (R10 media). During this time, lyophilized guinea pig complement (CL4051, Cedarlane) was reconstituted in 1 mL of cold water and centrifuged for 5 min at 4 °C to remove aggregates. Cells were washed with PBS and resuspended in 200 μL of guinea pig complement, which was prepared at a 1:50 dilution in Gelatin Veronal Buffer with Ca2+ and Mg2+ (IBB-300X, Boston BioProducts). After incubation at 37 °C for 20 min, cells were washed in PBS 15 mM (ethylenedinitrilo)tetraacetic acid (ThermoFisher Scientific) and stained with an anti-Guinea Pig Complement C3 FITC (polyclonal, ThermoFisher Scientific). Cells were then fixed with 4% formaldehyde solution, and fluorescence was evaluated on an LSRII flow cytometer (BD Bioscience).
Cl4051
Manufactured by Cedarlane
Sourced in Canada
The CL4051 is a laboratory equipment product from Cedarlane. It is a precision instrument designed for specific laboratory applications. The core function of the CL4051 is to perform accurate measurements and analysis within a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Anti guinea pig complement c3 fitc, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Neutravidin 1 μm beads, by Thermo Fisher Scientific (1 mentions)
Ca2 and mg2, by Boston BioProducts (1 mentions)
Fluorescein conjugated goat anti guinea pig complement c3, by MP Biomedicals (1 mentions)
Gelatin veronal buffer, by Merck Group (1 mentions)
Formaldehyde solution, by Tousimis (1 mentions)
Ca2 and mg2, by Merck Group (1 mentions)
3 protocols using cl4051
1
SARS-CoV-2 Spike Protein Neutralization Assay
2
Antigen-Dependent Complement Deposition Assay
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The ADCD assay was adapted from Fischinger et al. (57 (link)). Antigen was coupled to red fluorescent Neutravidin 1 μm beads (Invitrogen, Thermo Fisher Scientific, F8775) as described for ADCP. Immune complexes were formed by incubating 10 μL of coupled beads with 50 μL of antibody at concentrations of 50 μg/mL, 10 μg/mL, 2 μg/mL, and 0.4 μg/mL for 2 hours at 37°C. Plated were spun down, and immune complexes were washed with PBS. Lyophilized guinea pig complement (Cedarlane, CL4051) was resuspended in 1 mL of cold water, diluted 1:50 in GVB++ (gelatin veronal buffer and additional Ca2+ and Mg2+, Boston BioProducts, IBB-300X), and added to the immune complexes. The plates were incubated for 20 minutes at 37°C, and the reaction was stopped by washing the plates twice with 15 mM EDTA in PBS. To detect complement deposition, plates were incubated with fluorescein-conjugated goat anti–guinea pig complement C3 (MP Biomedicals, 0855385) for 15 minutes in the dark. Fluorescence was acquired with an Intellicyt iQue.
3
Complement-Mediated Viral Neutralization Assay
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This assay was described previously.27 Briefly, 293F-Spike-Delta cells, described above, were incubated with 100 μl of diluted plasma (10-fold) for 30 min at 4 °C. Cells were washed twice and resuspended in R10 media. Lyophilized guinea pig complement (CL4051, Cedarlane, Burlington, Canada, cat. no. CL4051) was reconstituted per the manufacturer’s instructions in 1 mL cold water and centrifuged to remove aggregates for 5 min at 4 °C. Cells were washed with PBS and resuspended in 200 μl of guinea pig complement, which was prepared at a 1:50 dilution in Gelatin Veronal Buffer with Ca2+ and Mg2+ (Sigma-Aldrich, St. Louis, MO, USA, cat. no. G6514). After incubation at 37 °C for 20 min, cells were washed in PBS 15 mM EDTA (ThermoFisher Scientific Baltics UAB, Vilnius, Lithuania, cat. no. AM9260G) and stained with an anti-guinea pig complement C3 FITC (polyclonal, MPBiomedicals, Solon, OH, USA, cat. no. 0855385). Cells were fixed with 4% formaldehyde solution (Tousimis, Rockville, MD, USA, cat. no. 1008B) and fluorescence was evaluated on a LSRII (BD Bioscience).
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