Fluorospheres

Bacterial distribution in human milk was determined after analyzing 10 ml of colostrum (n = 9) and mature milk (n = 9) samples, using a MoFloXDP cytometer with sorter. Transition samples were not analyzed due to lack of volume availability. Light was produced by an argon laser of 400 nm (blue light) and 200 Mw. First, the machine was calibrated using electromagnetic beads (Fluorospheres, Beckman Coulter Inc.) with known size (1, 3, and 10 μm). Then, events under 3 μm (containing planktonic bacteria) and those over 3 μm (containing human cells) were counted and sorted in two different tubes. DNA was extracted from both fractions for each sample, and qPCR was performed to determine the number of bacteria present in each of them, corresponding to free-living and human cells-associated bacteria. Fluorescence microscopy was performed on a selected number of samples after marking with DAPI dye, and visualized on a Nikon Eclipse E800 microscope. For Scanning Electron Microscopy, samples were kept on Karnovsky solution and further fixed with 1% OsO₄ in PBS buffer. Samples were then dehydrated with ethanol and critical-point drying, attached to a stub and coated with gold. Images were obtained in a Hitachi S-4800 Scanning electron Microscope with default settings at University of Valencia.

Non-biodegradable, fluorescent polystyrene microspheres of 10-µm diameter (Fluorospheres, Beckman Coulter, USA; light emission of 525–700 nm wavelengths when excited at 488 nm) were used as cell surrogates. Particle size was chosen considering regular human cell sizes. Aliquots of 5.0×10⁵ particles were prepared in 15 µl of Phosphate buffered saline (PBS) for each injection.

Blood was centrifuged and serum was stored in -80 °C until assayed. Levels of biomarkers were quantified by using commercially available kits of enzyme immunosorbent assays from Bio-Techne (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The lower limits of detection were: 16 pg/ml for tumor necrosis factor-alpha (TNFα); 40 pg/ml for IL-6; 31 pg/ml for IL-10; 62 pg/ml for IL-38; 156 pg/ml for IFNγ; and 313 p/ml for platelet-derived growth factor (PDGF)-A.
White blood cells were incubated for 15 minutes in the dark with the monoclonal antibodies anti-CD14 FITC, anti-HLA-DR-PE, anti-CD45 PC5 (Beckman Coulter, Marseille, France). White blood cells were also incubated for 15 minutes in the dark with anti-CD3 FITC, anti-CD4 FITC and anti-CD19 FITC (fluorescein isothiocyanate, emission 525nm, Beckman Coulter); with anti-CD4 PE, anti-CD8 PE, and anti-CD(16+56) PE (phycoerythrin, emission 575nm, Beckman Coulter); and with anti-CD45 PC5 (emission 667nm, Beckman Coulter). Fluorospheres (Beckman Coulter) were used for the determination of absolute counts. Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida). Isotypic IgG controls stained also with anti-CD45 were used for each patient. Results of HLA-DR on CD14/CD45-cells were expressed as mean fluorescence intensity (MFI).