Recombinant human c1q

HA was kindly provided by Prof. Ivan Donati (Department of Life Sciences, University of Trieste, Italy). The following antibodies were used: mouse monoclonal anti-lysosomal-associated membrane protein 1 (LAMP1) and mouse monoclonal anti-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-early endosome antigen 1 (EEA1), mouse monoclonal anti-calnexin, mouse monoclonal anti-mannose 6-phosphate receptor (M6PR), anti-mouse Alexa Fluor 594-conjugated antibody, anti-rabbit Alexa Fluor 488-conjugated antibody, rabbit polyclonal anti-HYAL1 (#PA5-79420), rabbit polyclonal anti-HYAL2 (#PA5-24223) and mouse monoclonal anti-CD44 (#MA513890) from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA); rabbit polyclonal anti-HYAL2 (#15115-1-AP) from Proteintech (Proteintech, Rosemount, IL, USA); donkey anti-rabbit HRP-conjugated secondary antibody from Sigma (Sigma-Aldrich, Saint Louis, MO, USA); anti-mouse LI-COR IRDye 680RD and anti-rabbit LI-COR IRDye 800CW from LI-COR Biosciences (LI-COR Biosciences, Lincoln, NE, USA); mouse monoclonal anti-gC1qR 74.5.2, rabbit polyclonal anti-gC1qR and rabbit polyclonal F(ab’)₂ anti-gC1qR were obtained as described earlier (28 (link)).
Recombinant human C1q was purchased from Sigma-Aldrich (#C1740). All other chemicals were purchased from Sigma-Merck.

To investigate the binding of C1q to pneumococci, bacteria were grown in THY broth to the early exponential phase (OD_600nm ~ 0.3), washed in TBS, and distributed in 96‐well plates (5 × 10⁷ CFU/well). Afterward, wells were blocked with TBS‐2% milk and incubated with the indicated doses of human recombinant C1q (Calbiochem; 204,876) for 1 h at 37°C. Cell‐surface bound C1q was revealed with a mouse anti‐C1q monoclonal antibody (Abcam; ab71940) diluted 1:100 in TBS‐1% milk followed by incubation with AP‐conjugated anti‐mouse IgG (Sigma‐Aldrich; A7434).
For flow cytometry immunofluorescence analysis, bacteria were incubated with recombinant human C1q (5 μg/ml) for 1 h at room temperature in constant agitation. Washed bacteria were incubated with anti‐C1q antibody (Abcam; ab71940) diluted 1:100 in 1% milk‐PBS for 1 h at room temperature in constant agitation. Afterward, to detect the binding of C1q to the bacteria, an Alexa‐Fluor‐488‐labeled anti‐mouse antibody was used. Bacteria were analyzed with a Flow Cytometer (BD FACS canto II) using the Flowlogic software.