C1q depleted human serum

Manufactured by Quidel

C1q-depleted human serum is a lab equipment product that contains human serum with the C1q component removed. C1q is a protein involved in the classical pathway of the complement system. The product is intended for use in research applications.

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Lab products found in correlation

Phenol free rpmi medium, by Thermo Fisher Scientific (2 mentions) Facscanto 2, by BD (1 mentions) 50 mpr microplate reader, by Agilent Technologies (1 mentions) Spectramax i3x plate reader, by Molecular Devices (1 mentions)

4 protocols using c1q depleted human serum

Removing IgG from Serum Using S. aureus

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To remove IgG from serum, S. aureus was added to a final OD₆₀₀ of 1.0 to NHS and incubated on ice for 30 min. The sample was centrifuged, and the pellet was discarded. Fresh S. aureus was added, and the serum was again incubated on ice for 30 min. After centrifugation, the supernatant was used as “preadsorbed serum.” S. aureus Newman Δspa was grown in phenol-free RPMI medium (Gibco) to an OD₆₀₀ ~1.0, washed 1x with PBS and resuspended in 3% BSA in PBS. Bacteria blocked for 30 min at room temperature (RT) and washed 2x with PBS were resuspended in 0.05% BSA in phenol-free RPMI to an OD₆₀₀ ~ 1. 200 μl of bacteria was mixed with 200 μl of NHS (Quidel), preadsorbed serum, C1q-depleted human serum (Quidel), or media plus antibody at 100 μg/mL final concentrations and incubated for 1 h at 37 °C with shaking. After incubation, samples were washed 2x with 1% BSA in PBS and resuspended in 1% BSA in PBS containing 3 μg/ml anti-C3b antibody (clone 3E7; Biolegend). After incubating for 1 h at RT and washing 2x with 1% BSA in PBS, samples were resuspended in 1% BSA in PBS containing 2 μg/ml goat anti-mouse IgG antibody, Alexa Fluor 488 conjugated (Invitrogen) and incubated at RT for 45 min. 0.5 μM ToPro3 (ThermoFisher) was added for 15 min. Samples washed 2x with 1% BSA in PBS were resuspended in 1% BSA in PBS and analyzed on a BD FACSCanto II.

Cruz J.W., Damko E., Modi B., Tu N., Meagher K., Voronina V., Gartner H., Ehrlich G., Rafique A., Babb R., Aneja P., Potocky T.B., D’ Orvilliers A., Coppi A., E S.Y., Qiu H., Williams C.M., Bennett B.L., Chen G., Macdonald L., Olson W., Lin J.C., Stahl N., Murphy A.J., Kyratsous C.A, & Prasad B.C. (2019). A novel bispecific antibody platform to direct complement activity for efficient lysis of target cells. Scientific Reports, 9, 12031.

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Assessing Complement Activation on HMEC-1 Cells

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HMEC-1 cells were cultured and incubated with a mAb W6/32. Cells were subsequently washed with PBS and incubated with culture medium with or without 10% NHS, C1q-depleted human serum (Quidel), C4-depleted human serum (Quidel), or NHS with addition of an inhibitor of factor B (fB, mAb 1379 described previously in (Thurman et al., 2005 (link))) or an inhibitor of factor I (fI, mAb A247, Quidel) at 37°C for one hour.
Complement inhibited serum was prepared by adding either mAb 1379 (40μg/mL) or mAb A247 (11μg/mL) to NHS. Preparations were incubated on ice for 30 minutes prior to incubation with cultured cells (Margaret A Lindorfer et al., 2013 ; Thurman et al., 2005 (link)). For these experiments, control NHS was also incubated on ice for 30 minutes prior to incubation with cultured cells to control for complement turnover in solution. Complement fragments were detected with the antibodies described above and fluorescence was measured on a FACSCalibur flow cytometer.

Stites E., Renner B., Laskowski J., Quintrec M.L., You Z., Freed B., Cooper J., Jalal D, & Thurman J.M. (2019). Complement Fragments are Biomarkers of Antibody-Mediated Endothelial Injury. Molecular immunology, 118, 142-152.

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Complement Pathway Modulation by Mirolysin

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To assess activity of the classical pathway, sheep erythrocytes were washed three times with DGVB⁺⁺ buffer (2.5 mM veronal buffer pH 7.3, 70 mM NaCl, 140 mM glucose, 0.1 % gelatin, 1 mM MgCl₂ and 5 mM CaCl₂). The cells were incubated with a complement-fixing antibody (amboceptor; Boehringwerke; diluted 1:3000 in DGVB⁺⁺ buffer) at a concentration of 10⁹ cells/ml for 20 min at 37°C. After two washes with DGVB⁺⁺, 5×10⁸ cells/ml were incubated for 1 h at 37°C with 0.3% NHS diluted in DGVB⁺⁺ buffer (total volume 150 μl). Before incubation with erythrocytes, NHS was pre-incubated with various concentrations of mirolysin for 15 min at 37°C. The samples were centrifuged and the amount of lysed erythrocytes was determined by spectrophotometric measurement of the amount of released hemoglobin (405 nm; Varian 50 MPR Microplate Reader).
To assess activity of the alternative pathway, rabbit erythrocytes were washed three times with GVB⁺⁺ buffer and then used at a concentration of 5×10⁸ cells/ml for 1 h incubation at 37°C with 3% C1q-depleted human serum (Quidel) diluted in GVB⁺⁺ buffer (total volume 150 μl). The serum used was pre-treated with mirolysin variants for 25 min at 37°C. The samples were centrifuged and the amount of lysed erythrocytes was determined spectrophotometrically (405 nm; Varian 50 MPR Microplate Reader).

Jusko M., Potempa J., Mizgalska D., Bielecka E., Ksiazek M., Riesbeck K., Garred P., Eick S, & Blom A.M. (2015). A metalloproteinase mirolysin of Tannerella forsythia inhibits all pathways of the complement system. Journal of immunology (Baltimore, Md. : 1950), 195(5), 2231-2240.

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Serum-mediated Killing of S. aureus

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S. aureus Newman Δspa was grown in phenol-free RPMI medium (Gibco) until it reached an OD₆₀₀ ~1.0, washed once with PBS and resuspended in 3% BSA in PBS. They were blocked for 30 min at room temperature and then washed two times with PBS and resuspended in 0.05% BSA in phenol-free RPMI to an OD₆₀₀ ~ 1. 200 μl of bacteria was mixed with 200 μl of NHS (Quidel), 200 μl C1q-depleted human serum (Quidel) or C5-depleted serum (Quidel) and samples were incubated for 0, 10 or 24 h at 37 °C with shaking. After incubation, propidium iodide (PI) was added to each sample to a final concentration of 1 μg/ml and incubated at room temperature for 5 min. Fluorescence was measured using a SpectraMax i3x plate reader (excitation 533 nm, emission 617 nm; Molecular Devices). Separate samples were prepared in triplicate for 0, 10 and 24 h analysis and results are plotted as mean with standard deviation.

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