Fura 2 am

Pre-B 300.19-CXCR4⁺ cells (0.2 × 10⁶) in 200 µl were loaded with FURA 2-AM (final concentration 1 µM) on poly-d-lysine–coated slides (MatTek, Ashland, MA, USA). Loaded cells were washed with buffer that contained 136 mM NaCl, 4.8 mM KCl, 20 mM HEPES, 1 mM CaCl₂, and 10 mM glucose. Imaging was recorded with a ×40 oil-immersion objective on an inverted microscope (Axiovert 200; Carl Zeiss, Jena, Germany) with excitation at 340 and 380 nm using the Polychrom V illumination system from Till Photonics GmbH (Munich, Germany). Chemokine was injected after 50 s of recording, and recording continued up to 300 s. The 340/380 ratio provides a relative measure of cytoplasmic free Ca²⁺ concentration.

Intracellular calcium concentration ([Ca²⁺]i) was measured in single EA.hy926 cells using ratiometric calcium indicator Fura 2-AM (Life Technologies, Carlsbad, CA). Cells grown on 35 mm glass-bottom plates (MatTek, Ashland, MA) were loaded with 1 μM Fura 2-AM in calcium buffer (140 mM NaCl, 2.8 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose and 10 mM HEPES) for 15 min, washed and incubated in calcium buffer for another 15 min. Fura 2-AM-loaded cells were illuminated in calcium buffer by alternating 340/380-nm light delivered every 300 ms by a DG-4 argon exciter (Sutter Instruments, Novato, CA) under the control of MetaFluor software, and fluorescence images were captured at an emission of 510 nm with a Photometrics Cool SNAP HQ charge coupled device camera (Roper Scientific, Tucson, AZ) based on a Nikon TE2000 fluorescent microscope. Calf thymus histones (50 µg mL⁻¹) without or with 3 min of pre-incubation with RNA aptamer KU7 at various concentrations were added to loaded cells and calcium images were recorded for 10 min for each treatment condition. [Ca²⁺]i was reported as the ratio of Fura 2 fluorescence emission at 340 and 380 nm (F340/F380) normalized to baseline. All procedures were performed at room temperature.