Syto deep red nucleic acid stain

Isolated peripheral blood neutrophils stained with SYTO^TM Deep Red nucleic acid stain (Invitrogen) were stimulated with 20 μg/ml SLE-IC for 1.5 h and placed in culture. Z-stack images were taken at Day 0 and at Day 2 were collected using a DMI6000 (Leica) with a 63X NA1.3 Plan-Apo glycerol immersion objective, CSUX Yokogawa spinning disc (Andor), Borealis illumination system (Andor), and Zyla Plus camera (Andor), controlled by MetaMorph 7.8 (Molecular Devices).

The intracellular localization and co-localization of the formulations with lysosomes were evaluated by confocal microscopy. Cells were seeded at 5 × 10⁵ cells per ml onto Poly-D-Lysine coated coverslips in a 24-well plate and treated with FAM-labelled siRNA contatining formulations. The incubation periods varied from 6, 12 to 24 h. LysoTracker^TM Red DND-99 (Invitrogen, Invitrogen, Waltham, MA, USA), a red-fluorescent dye for labelling and tracking acidic organelles, was prepared at 100 nM in serum-free media. The Syto^TM Deep Red Nucleic Acid Stain (Invitrogen, Invitrogen, Waltham, MA, USA), which stains the nuclei of cells, was added to the Lysotracker working solution to give a final nucleic acid stain of 1 µM. After incubation, the media was removed carefully, and the dyes were added to each well (500 µL dye solution/well); after 150 min the dye solution was withdrawn. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature followed by wash with PBS. The coverslips were removed and mounted with Fluorescent Mounting Media (Dako, Agilent, Santa Clara, CA, USA); the cells were imaged using an OLYMPUS FV1000 Confocal Laser Scanning Biological Microscope.

Cells were grown on sterile cover slips placed in Petri dishes. After treatment, cells were fixed (4% paraformaldehyde), permeabilized (0.1% Triton X-100) and stained with 0.6 µM Phalloidin–Atto 565 (Sigma Aldrich Inc., St. Louis, MO, USA) and 1 µM SYTO Deep Red Nucleic Acid Stain (Thermo Fisher Scientific, Waltham, MA, USA). The samples were scanned using the laser confocal microscope Nikon Eclipse Ti-E A1R-Si and Nikon NIS Elements AR software.