Cd80 fitc

Anti-mCRP-9C9 and anti-pCRP-8D8 antibodies were purified from hybridoma culture supernatants58 (link). Anti-CRP antibody clone CRP-8, mouse IgG FITC-conjugated F(ab′)₂ and human IgG-purified immunoglobulins were purchased from Sigma-Aldrich. CD16-FITC, CD32-FITC, CD64-FITC, CD14-PE, CD68-PE, CD80-FITC, CD163-FITC and respective control antibodies were from Miltenyi Biotec (Bergisch Gladbach, Germany). Anti-ICAM-1 (#4915) and NF-κB pathway antibodies (NF-κB Pathway Sampler Kit; #9936) were from Cell Signaling Technology (Danvers, MA, USA). Anti-VCAM-1-horseradish peroxidase (anti-VCAM-1-HRP) (E-10) and anti-GAPDH-HRP (0411) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Complement C3-PE antibody (clone 6C9), which reacts with human C3 as well as the breakdown products C3b and iC3b, was purchased from LifeSpan BioSciences (Seattle, WA, USA) and Alexa Fluor 488 Annexin V from Life Technologies (Carlsbad, CA, USA). APC goat anti-mouse IgG was from BD Biosciences (San Diego, CA, USA). Anti-iC3b (neoepitope), anti-C3c (recognizes C3, C3b, iC3b, C3c) and anti-C3d (recognizes C3, C3b, iC3b, C3d) antibodies were purchased from Quidel (San Diego, CA, USA).

To analyze DC maturation, BMDC were stimulated for 24 h with E. coli-derived LPS (1 μg/ml) or heat killed B. microti (HKBM; 100 bacteria/cell). Subsequently, the expression of MHCII, CD40, CD86 and CD80 was analyzed on CD11c⁺ cells using MHCII-APC, CD40-APC, CD86-FITC, CD80-FITC and CD11c-PE antibodies (Miltenyi Biotec). Samples were fixed with 4% PFA and analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences).

Next, 24 h after LPS stimulation, BMDCs were detached from the plates with DPBS 1× (Gibco, MA, USA) + 0.5mM EDTA (Thermo Fisher Scientific, MA, USA). Cells were then washed with DPBS 1× + 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and labelled with CD11b VioBrightFITC (Miltenyi Biotec, Bergisch Gladbach, Germany), MHCII PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD11c PECy5 (Miltenyi Biotec, Bergisch Gladbach, Germany), F4/80 APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD80 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD86 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and 7-AAD PECy5 (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow Cytometer data analysis was performed using NAVIOS software (Beckman Coulter, Brea, CA, USA), with at least three experiments performed. Flow cytometer analysis was performed using Kaluza Software 1.5 (Beckman Coulter, Brea, CA, USA).