Dq red bsa

For degradation ability analysis, cells were loaded with DQ-Red BSA (20 μg/ml, Invitrogen) for 1 h at 37°C and then incubated with fresh culture medium without DQ-Red BSA for another 1 h. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) immediately. Imaging was performed with a Plan-APOCHROMAT 100×/1.4 Oil DIC oil immersion objective on an LSM980 confocal microscope (Zeiss).

The fluorogenic assays for phagosomal proteolysis and acidification were adapted from Russell laboratory (Yates et al, 2009 (link)). Cells were seeded into a 96 well plate at 1 × 10⁵ cells per ml 24 h prior to the experiment. Carboxylate silica beads (3 μm, Kisker Biotech) were conjugated with DQ red BSA (Molecular Probes) or pHrodo (Molecular Probes) and incubated for 3 min at 1:100 in binding buffer (1% FBS in PBS pH 7.5) with seeded macrophages at 37°C. Solution was replaced with warm binding buffer and cells were immediately measured at 37°C. Real‐time fluorescence was measured using a SpectraMax Gemini EM Fluorescence Microplate Reader (Molecular Devices), set as maximal readings per well to allow reading time intervals of 2 min. The excitation/emission wavelengths were 590/620 nm (DQ red BSA), 650/665 nm (Alexa Fluor 640), 560/585 nm (pHrodo) for the proteolysis or acidification assay and measured in relative fluorescence units (RFU). Plots were generated from the ratios of signal/control fluorescence. Error bars were generated from standard error of the mean of 6 replicates.

The bioactive Lf from bovine whey was kindly supplied by the Tatua Cooperative Dairy Company Ltd (New Zealand). Poly-L-Lysine hydrochloride (PLL, Mw 15,000–30,000), BSA lyophilized powder (≥96%), TA (ACS grade), BSA conjugated with fluorescein isothiocyanate (BSA/FITC), bile salts (for microbiology), pancreatin from porcine pancreas (≥100 USP U/mg), pepsin from porcine gastric mucosa (3802 U/mg), mini protease inhibitor cocktail cOmplete™, hydrochloric acid, calcium chloride dehydrate, sodium bicarbonate, sodium chloride, sodium hydroxide, acetonitrile (for HPLC, ≥99.9%) were purchased from Sigma-Aldrich. Anhydrous sodium carbonate was purchased from Alfa Aesar. 0.1% aqueous solution of trifluoroacetic acid (TFA, LC/MS grade, ≥99.99%) was purchased from VWR Chemicals. DQ™ Red BSA was purchased from Molecular Probes Inc, USA. The bovine lactoferrin ELISA kit was purchased from Bethyl Laboratories, Inc., USA. Cyanine 7 N-hydroxysuccinimide ester (Cy7-NHS) was purchased from Lumiprobe Corporation, USA. Cy7-labelled Lf (Cy7-Lf) has been prepared according to a standard protocol provided by Lumiprobe Co and purified by dialysis. All chemicals were used as received without further purification. Deionized (DI) water (specific resistivity higher than 18.2 MΩcm) from Milli-Q plus 185 (Millipore) water purification system was used to make all solutions.