Prolong gold solution

Cells were seeded in 10 mm³ plates, where three sterile 1 cm circular coverslips had been previously introduced. After cell treatment, each crystal was transferred to a well of a 24-well plate, where the remainder of the protocol was carried out. Cells were fixed with 3% paraformaldehyde in H₂O for 30 min at room temperature (RT). Then, cells were washed with 200 mM glycine solution for 15 min at RT and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 30 min at RT. Cells were blocked in 1% BSA 2X PBS solution for 30 min at RT. Subsequently, they were incubated with the γ-H2AX antibody 1:100 diluted in blocking solution overnight at 4 °C. Washes were made with 1X PBS for 5 min and incubated with the goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (ThermoFisher Scientific) diluted in blocking solution for 1 h at RT in the dark. After washing with 1X PBS, nuclei were stained with DAPI (Life Technologies, Carlsbad, CA, USA) diluted in PBS (1:1000 dilution) for 15 min RT in the dark. Crystals were mounted on the slides with ProLong Gold solution (Life Technologies). The slides were stored at 4 °C until analysis. Images were obtained with the Leica TCS-SP2-AOBS confocal microscope and analysed with LCS Lite and Fiji Software v1.8.0.

HBMECs treated with siRNAs were reseeded onto 8-well chamber slides at appropriate cell concentrations. After fixation with 4% paraformaldehyde and permeabilization with Triton X-100, the cells were blocked with 10% normal goat serum for 1 h at room temperature. Next, the cells were incubated with primary antibodies at 4 °C overnight and subsequently stained with Alexa-594 or Alexa-488 conjugated secondary antibodies (Invitrogen). The stained samples were mounted in Prolong Gold solution (Invitrogen), and images were captured using a Leica TCS SP5 confocal microscope.

Cells were rinsed with Phosphate Buffered Saline (PBS) and fixed with either 4% paraformaldehyde in PBS for 10 min at room temperature or with methanol for 10 min at 4°C. They were then permeabilized with 0.5% Triton-X 100 in PBS 3 times for 10 min. The same solution was used for all subsequent washing steps. Cells were incubated with primary antibodies for 1 h at 37°C. After incubation, cells were washed for 30 min and incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at 37°C, and nuclei were labeled with DAPI (0.1 ηg/ml in 0.9% NaCl). Cells were mounted in Prolong Gold solution (Invitrogene) and examined with an Axiovert 100 microscope (Carl Zeiss, Germany) or with a laser scanning confocal microscope (TCS SP5 AOBS, Leica, Japan). Image processing and stack projections were performed using Fiji software (based on ImageJ, http://imageJ.nih.gov/ij/). Control experiments with no primary antibodies showed only a faint background staining (data not shown). Some cultured cells were also examined under phase contrast microscopy with an Axiovert 100 microscope (Carl Zeiss, Germany).

Neutrophils were fixed in 4% paraformaldehyde in PBS overnight at 4°C, washed, and blocked with 0.2% porcine gelatin (MilliporeSigma) for 30 minutes, then incubated with primary Ab (anti-C1q, Dako, catalog a0136; or patient serum) for 1 hour in a humid chamber at 37°C. Coverslips were then washed 3 times and incubated 30 minutes with secondary Abs at 37°C: cit-H4 (MilliporeSigma, catalog 07-596), goat anti-rabbit IgG (Invitrogen, catalog A32731), and goat anti-human IgG (Invitrogen, catalog A-11013). Nuclei were counterstained with 1:1000 Hoechst at RT. After washing 3 more times, coverslips were mounted on glass slides using Prolong Gold solution (Invitrogen). Images were acquired on a Zeiss LSM 780 confocal microscope.