Celltracker green fluorescent dye

A human monocyte cell line from acute monocytic leukemia THP-1 (ATCC^® TIB-202^™) was used to mimic human WBCs. THP-1 cells were cultured following the manufacturer’s instruction. Briefly, THP-1 cells were cultured in RMPI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% v/v FBS (Gibco) and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich). Cells were maintained in an incubator at 37°C and 100% humidity. Culture medium was exchanged every 2 – 3 days, and cells were passaged when reaching confluence. For visualization and cell counting, THP-1 cells were pre-stained with CellTracker Green fluorescent dye (Molecular Probes, Grand Island, NY). CellTracker Green was directly added into culture medium to a final concentration of 1 μM and incubated with cells at 37°C for 10 min.
Cell viability was examined using Trypan Blue. Briefly, cell suspension was mixed with 0.4% Trypan Blue Stain (Gibco) at a ratio of 5:1. After 5 min of incubation at RT, cells were injected into a hemocytometer and observed under a phase contrast microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Cells that excluded the stain were classified as viable cells, whereas stained cells were identified as non-viable.