Pe conjugated anti mouse ab

3-(4,5-dimethylthiazol-diphenyltetrazolium bromide (MTT), was purchased from Sigma Aldrich (Milan, Italy). The following rabbit polyclonal antibodies (Abs) were used: Anti-histone H3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-COX IV (1:250; Cell Signaling Technology) and anti-caveolin-1 (1:400; Cell Signaling Technology). The following mouse monoclonal Abs were used: anti-TRPML1 (clone F-10, 1:300 for Western blot, 1:50 for FACS analysis; Santa Cruz Biotechnology, Dallas, TX, USA), anti-TRPML2 (1:300 for Western blot; Santa Cruz Biotechnology), anti-LAMP1 (1:300; Santa Cruz Biotechnology), anti-calreticulin (1:1000; BD Biosciences, Milan, Italy) and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, 14C10, 1:1000; Cell Signaling Technology). anti-TRPML2 from Sigma Aldrich was used diluted 1:50 for FACS analysis.
The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG (1:2000; Cell Signaling Technology), FITC-conjugated anti-rabbit Ab (BD Biosciences), PE-conjugated anti-mouse Ab (BD Biosciences, Milan, Italy), Alexa Fluor-594-conjugated anti-mouse Ab (1:100; Invitrogen, San Diego, CA, USA), Alexa Fluor-488-conjugated anti-mouse Ab (1:100; Invitrogen) and Alexa Fluor-488-conjugated anti-rabbit Ab (1:100; Invitrogen).

Gelatin veronal buffer (GVB, Sigma-Aldrich, St. Louis, USA) supplemented with 13.3% ΔC1f2 serum and 16.6% CEMf2 was preincubated with or without inhibitors on ice for 15 min. 75µL of prechilled mixture was added to 25µL of GVB containing 5 × 105 CFUs of bacteria in microplate wells. Plates were incubated for 30 min at 37°C in 5% CO2 with shaking and then placed on ice to stop complement deposition. After bacteria were washed with ice-cold GVB, surface bound C3 or C4b/C4c bound were stained using murine anti-human C3 mAb (ThermoFisher Scientific, Waltham, USA; LF-MA0132, clone 28A1) and PE-conjugated anti-mouse Ab (BD Biosciences, Franklin Lakes, USA), or FITC-conjugated murine anti-human C4 mAb (ThermoFisher Scientific, Walktham, USA), and detected by flow cytometry using BD Accuri C6 Plus (BD Biosciences, Franklin Lakes, USA) and FCS Express software (Pasadena, USA).