Las x imaging software

For live cell experiments, cultured cells were imaged using an Olympus fluorescence microscope (Waltham, MA), or a Leica TCS SP8 confocal microscope (Buffalo Grove, IL). Images were taken at indicated time-points using the same exposure conditions within the group for comparison. To show the intracellular localization of the fluorescent fusion proteins, both fluorescence and phase/TLD images were overlaid using the Leica Las X imaging software.

Cells were seeded at 250,000 cells per mL in 4 mL and treated with WGA. Cells were spun at 600 rpm for 5 min and washed with PBS twice. Pellet was resuspended in PBS and vortexed to make single cell suspension. While vortexing the sample, 1 mL of ice-cold 70% ethanol was added. Samples were incubated overnight in −20°C. Then, samples were pelleted, washed, resuspended in PBS, and incubated with 100 μL of Propidium Iodide at room temperature for 15 min. Samples were analyzed with FACS, counting 10,000 events. Events collected were gated on live cell populations, avoiding debris and aggregate populations.
For cell aggregation/agglutination assay, HL-60, OCI, and healthy human white blood cells (WBCs) were seeded in 12-well plates at a concentration of 250,000 cells/mL (1 mL per well). Cells were treated with either 2 μg/mL WGA or with 2 μL PBS as a negative control. After 20 h treatment, cells were assessed at 10x magnification using bright field microscopy (Leica DM IL LED) and captured using Leica LAS X imaging software.

The attached cells in the glass-based Petri dishes were covered with PBS with 1 mM Ca²⁺/0.5 mM Mg²⁺ and imaged using a confocal microscope Leica TCS SP5 (Leica, Wetzlar, Germany). Na,K-ATPase was labeled with FITC-conjugated antibodies and imaged using a 488 nm argon laser. Aβ₄₂ was labeled with Texas Red-conjugated antibodies and imaged using a 594 nm HeNe laser. The resulting images were analyzed using LAS X imaging software (Leica, Wetzlar, Germany).