Micro chemotaxis boyden chamber

MOVAS were incubated for 24–48 h in the presence or absence of UA 9 mg/dL and/or Probenecid, Losartan, Valsartan and MG-132. Then, cells were tested for migration in a micro-chemotaxis Boyden chamber (Neuro Probe Inc., Gaithersburg, MD, USA) using a 8 μm-pore size, polycarbonate polyvinylpyrrolidone-free filters (Millipore). The lower wells of the chemotaxis chambers were filled with medium alone. After incubation (3 h, 37 °C), the filters were removed from the chambers, fixed and stained with May Grunwald–Giemsa stain (Carlo Erba, Cornaredo, Italy). Each condition was performed in duplicate. The cells of five random oil-immersion fields were counted and the chemotaxis index was calculated from the number of cells migrated to the test samples divided by the number of cells migrated to the control [12 (link)].

6T-CEM cells were transfected with lncRNA ENST00000502883.1 siRNA or scrambled siRNA for 48 h, the supernatant was collected for chemotaxis analysis. The PBMCs were isolated and suspended at a concentration of 10⁶ cells/ml in chemotaxis buffer (RPMI 1640 containing 25 mM Hepes and 1% (v/w) endotoxin-free bovine serum albumin). The chemotaxis protocol was performed using a 48-well microchemotaxis Boyden chamber (Neuro Probe, Cabin John, MD) with 5 μm pore polycarbonate filters (Neuro Probe). The inferior wells were loaded with cell culture supernatants pretreated at 37°C for 30 min with neutralizing Ab against CXCL16 (R&D systems, Minneapolis, MN, USA) or goat IgG isotype control (R&D systems), chemotaxis buffer, and CXCL12 (PeproTech) at 10⁻⁷ M were used as negative and positive controls, respectively. 20 ng/ml of CXCL16 (PeproTech) pretreated with neutralizing Ab against CXCL16 (R&D systems) or goat IgG isotype control (R&D systems) was loaded into the inferior wells as well. The chemotaxis system was conducted for 2 h 30 min at 37°C in 5% CO₂. Each condition was performed in triplicate. Cells having migrated through the filter were counted in the inferior well, and results were expressed as index of chemotaxis compared with chemotaxis buffer.