Diff quik set

THP-1 monocytes or purified peritoneal macrophages were primed with HG+LDL for 20–24 h in the presence of either vehicle (dimethyl sulfoxide, DMSO, ≤0.1%) or UA, then loaded into the upper wells of a 48-well modified Boyden chamber (NeuroProbe, Gaithersburg, MD). The lower wells contained either vehicle or 2 nM MCP-1 (R&D Systems, Minneapolis, MN). A 5 µm polyvinyl pyrrolidone-free polycarbonate filter membrane was layered between the upper and lower chambers, and the chamber was incubated for 2 h for THP-1 monocytes or 3 h for peritoneal macrophages at 37 °C and 5% CO₂. The membrane was washed and cells removed from the upper side of the filter. Transmigrated cells were stained with Diff-Quik^® Set (Dade Behring, Newark, DE) and counted in four–five separate high power fields at 400× magnification under a light microscope.

Cells were pre-incubated with DMSO or PLTFBH for 12 h. The cells were then split, and viable cells were counted by trypan-blue staining. Viable cells (1–4 × 10⁴) suspended in DMEM with 0.5% FBS containing either DMSO or PLTFBH were placed in the upper permeable cell culture inserts (24-well plate, 8.0 µm pore size, CELLTREAT Scientific Products, Pepperell, MA, USA). The bottom chamber containing DMEM with 10% FBS was used as a chemoattractant. After 12 h, migrating cells on the bottom of the membrane were fixed, permeabilized, and stained with Diff-Quik Set (Dade Behring, Deerfield, IL, USA). Migrating cells were viewed using the EVOS M5000 microscope (Thermo Fisher Scientific). The numbers of migrating cells in the entire field were counted, and the percentage of the migrating cells was calculated compared to the control group.