Transwell inserts with polycarbonate membranes

The chemotaxis of murine and human CD8+ T cells was assayed in 24-well plates (5-μm-pore-size Transwell inserts with polycarbonate membranes; Corning). Medium alone (RPMI 1640 plus 10% FBS) or macrophage culture supernatants were added to the bottom compartments of triplicate wells. Murine or human T-cell migration was assessed with medium, APG-2575-treated supernatant alone, supernatant plus 10 μg/mL anti-CCL5 neutralizing antibodies, and/or 10 μg/mL anti-CXCL10 neutralizing antibody (R&D Systems). CD8+ T cells (5 × 10⁵) from C57BL/6 mouse spleens or the blood of healthy donors were purified with mouse or human anti-CD8a beads (Miltenyi Biotec), placed in Transwell inserts and incubated at 37 °C for 8–12 h. Cells in the bottom compartments were enumerated by flow cytometry. The numbers of spontaneously migrated cells were subtracted from the total number of migrated cells under all conditions, and the data are reported as the chemotactic index. Chemotactic index = (migrated cells – spontaneously migrated cells)/total T cells plated in the Transwell insert × 100%.

All leukemia cell lines employed were rendered quiescent by incubation in RPMI medium supplemented with 0.5% bovine serum albumin (BSA, Sigma-Aldrich) at 37°C and then seeded at a density of 10 × 10⁴ cells/well into the upper chambers of Transwell inserts with polycarbonate membranes and 8–μm pore size (Corning). The lower Boyden chambers received different concentrations of SexHs into RPMI medium with 0.5% BSA. The pituitary hormones were purchased from ProSpec (East Brunswick, NJ, USA), while the gonadal hormones were purchased from Sigma-Aldrich. The lower chambers containing stromal-derived factor 1 (SDF-1; PeproTech) and 0.5% BSA in RPMI 1640 medium were served as a positive and negative control, respectively. After 3 h of stimulation at 37°C incubation, the upper chambers were carefully removed, and the cells that had migrated to the lower chambers were collected and scored using FACS analysis.