Chemotaxis buffer

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.

TCRβ^−ve NK1.1⁺ CD11b⁺ KLRG1⁺ NK cells were resuspended in chemotaxis buffer (RPMI 1640 and 0.5% BSA). Approximately 5 × 10⁵ cells were placed in the upper chambers of 24-well transwell plates (3-μm filters; Corning) above 600 μl of chemotaxis buffer with or without chemokine (PeproTech) and incubated at 37°C and 5% CO₂ for 3 h. Migrated cells were enumerated by FACS using CountBrite beads (Invitrogen) and converted into the percentage of input for each subset.