In our imaging system, fluorescently labeled cells suspended in a HARV-type RWV were irradiated by a laser-beam.
For relaxation time measurements, 1 × 105 labeled cells were suspended in gelatin (2%, 500 μL) in plastic vials homogenously.
The labeled samples were suspended in hybridization buffer and applied to the array slides.
The labeled NSCs were suspended in serum-free Opti-MEM and seeded into the Boyden chamber Transwell inserts at a cell density of 5 × 10 per insert.
PA (live and heat-killed) was labeled with FITC, suspended in assay buffer (20 mM HEPES, 150 mM NaCl, 1% BSA, pH 7.4) and enumerated using a bacterial cytometer, a fluorescent microscope and by flow cytometry (BD-FACS Calibur) [6].
Single-cell suspensions (1 × 10) isolated from lungs of immunized mice obtained 30 minutes, 2, 4 and 8 hours after the injection of labeled mRNA were suspended in RPMI 1640 medium (Life Technologies, Grand Island, NY).
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In either case, the cells and labeling agents were suspended in binding buffer (200 μl) prepared by supplementing PBS with 4.5 g/L of glucose and 5 mM of MgCl2.
Prior to labeling, cells were suspended in PBS containing 1.26 mM CaCl2, 0.81 mM MgSO4 and 5 mM EDTA (Buffer A).
Roughly 1 × 10 labelled neutrophils were suspended in HBSS and added to a fibronectin-coated, chambered coverglass slide, which was maintained at 37 °C.
