C5 depleted serum

Manufactured by Complement Technology

Sourced in United States

C5-depleted serum is a laboratory reagent that has been processed to remove the C5 component of the complement system. The complement system is a part of the immune response, and C5 plays a role in this process. The removal of C5 results in a serum preparation that lacks this specific protein.

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Lab products found in correlation

Ecl substrate, by Promega (1 mentions) Pierce nhs activated magnetic beads, by Thermo Fisher Scientific (1 mentions)

6 protocols using c5 depleted serum

Bacterial Uptake by Neutrophils

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This assay was carried out as previously described (71 (link)). Briefly, GFP-producing NR3055 (NR3055 pJH026) and GFP-producing NR3055 SR2 (NR3055 SR2 pJH026) were opsonized with 2% C5-depleted serum (Complement Technology, Inc., Tyler TX) for 30 min at 37°C. Purified human PMNs were infected with each strain at an MOI of 10. The samples were incubated at 37°C in a roller drum incubator and at 0 hpi, 0.5 hpi, 1 hpi, 2 hpi, and 3 hpi, the fluorescence was analyzed via flow cytometry to detect the bacterial uptake through gating for PMNs by forward versus side scatter. The pJH026 vector (72 (link)), which contained gfp driven by the P_em7 promoter, was introduced into NR3055 and NR3055 SR2 via electroporation, as described above.

St. John A., Perault A.I., Giacometti S.I., Sommerfield A.G., DuMont A.L., Lacey K.A., Zheng X., Sproch J., Petzold C., Dancel-Manning K., Gonzalez S., Annavajhala M., Beckford C., Zeitouni N., Liang F.X., van Bakel H., Shopsin B., Uhlemann A.C., Pironti A, & Torres V.J. (2023). Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei. mBio, 14(2), e02590-22.

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Complement Protein Isolation Protocol

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Normal human serum was obtained from a pool of healthy donors as previously described (46 (link)). C5-depleted serum was obtained from Complement Technology. Complement protein C3 was isolated and purified from freshly obtained human plasma, as described before (47 (link)). Complement protein C4 was obtained from Complement Technology. Complement proteins C5 WT (unless stated differently) and C5 R885H and complement inhibitor RaCI3 were recombinantly produced and purified in our lab, using Expi293F cells.

Struijf E.M., De la O Becerra K.I., Ruyken M., de Haas C.J., van Oosterom F., Siere D.Y., van Keulen J.E., Heesterbeek D.A., Dolk E., Heukers R., Bardoel B.W., Gros P, & Rooijakkers S.H. (2023). Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies. The Journal of Biological Chemistry, 299(8), 104956.

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Bacterial Opsonization by Complement C5

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To generate pre-opsonized bacteria, E. coli MG1655 were grown to midlog phase (OD₆₆₀ ~ 0.5) in lysogeny broth (LB) and suspended in VBS containing 0.1 % BSA and 2.5 mM MgCl₂ (VBS⁺). Bacteria (~5 × 10⁸ cfu/ml) were pre-incubated for 1 h at 37 °C in 10 % C5 depleted serum (Complement Technology, Inc.), or 10 % normal serum that was heated at 56 °C for 30 min to eliminate complement activity as a control (HI serum). To allow decay of Bb, bacteria were incubated in PBS for 1.5 h at 37 °C. Then, bacteria were washed in VBS⁺ and incubated with 20 μg/ml C5 for 1 h at 37 °C under shaking conditions. After subsequent washing, binding of C5 was detected by flow cytometry. Bacterial cells (total of 10,000 events) were gated based on forward/side scatter and the geometric mean fluorescence of the gated population was analyzed using FlowJo software.

Berends E.T., Gorham RD J.r., Ruyken M., Soppe J.A., Orhan H., Aerts P.C., de Haas C.J., Gros P, & Rooijakkers S.H. (2015). Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes. BMC Biology, 13, 93.

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Measuring C3b Deposition on Bacterial Cells

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NR3055, NR3055 SR2, and NR3055::wzy cell suspensions in PBS at an OD₆₀₀ of approximately 0.1 were incubated with 2% C5-depleted serum (Complement Technology, Inc., Tyler, TX), 2% heat-inactivated C5-depleted serum, or no serum for 30 min at 37°C. The bacteria were washed once with RPMI and were then incubated with 3 μg/mL purified FITC anti-complement C3b/iC3b Antibody (BioLegend, San Diego, CA) for 30 min at 4°C while rocking. The fluorescence was analyzed via flow cytometry to measure the binding of antibodies to the C3b bound bacteria.

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Complement-mediated Cytotoxicity Assay

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CP convertase assay was performed as described in [17 (link)], with further modifications described in [18 (link)]. Briefly, cells were loaded with calcein AM, then pelleted in a V-shaped plate, overlaid with 25 µL of PBS, and incubated at 37 °C with shaking at 600 rpm. A mixture of ofatumumab (100 µg/mL) and 20% of C5-depleted serum (Complement Technology) (Tyler, TX, USA) supplemented with 5 µg/mL of respective C2 variant (all in a total volume of 25 µL) were added at a specific time point. Then, cells were washed with EDTA-GVB buffer (40 mM EDTA, 5 mM veronal buffer, 0.1% gelatin, 145 mM NaCl), pelleted, and overlaid with EDTA-GVB buffer containing 1:20 dilution of guinea pig serum (Harlan Laboratories) (Indianapolis, IN, USA). The readout of calcein release was performed as in the CDC assay.

Urban A., Majeranowski A., Stasiłojć G., Koszałka P., Felberg A., Taszner M., Zaucha J.M, & Okrój M. (2022). In Silico Designed Gain-of-Function Variants of Complement C2 Support Cytocidal Activity of Anticancer Monoclonal Antibodies. Cancers, 14(5), 1270.

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Affinity Purification of Complement Proteins

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RaCI2 and OmCI at 1 mg/ml in PBS were coupled to Pierce NHS-Activated magnetic beads (Thermo Scientific) following manufacturer’s instructions. 20 μl beads were incubated with human serum in the presence of 10 mM EDTA, for 30 minutes at room temperature. Beads were washed three times with 1 ml PBS supplemented with 0.005% Tween-20 and once with 100 μl PBS. Bound proteins were recovered by boiling the beads in non-reducing SDS-PAGE loading buffer, prior to separation on SDS-PAGE gel and analysis by western blot. C5-depleted serum (Complement Technology, USA) was included as a control. Western blots were incubated with 1/2,000 diluted polyclonal anti-C5 antibodies (Calbiochem) followed by 1/2,500 diluted anti-rabbit HRP (Promega). C3 and C4 were detected with anti-C3 (1/50,000) and anti-C4 (1/150,000) polyclonal antibodies (both from Complement Technology, USA) and anti-goat HRP antibodies (1/10,000) (Promega). Blots were incubated with ECL substrate (Promega) and imaged with film.

Jore M.M., Johnson S., Sheppard D., Barber N.M., Li Y.I., Nunn M.A., Elmlund H, & Lea S.M. (2016). Structural basis for therapeutic inhibition of complement C5. Nature structural & molecular biology, 23(5), 378-386.

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