Hcx apo l

Immature DCs (1×10⁵/ml) were cultivated on poly-L-lysine (PLL, 10 µg/ml, Sigma) pre-treated cover-slips, washed with Krebs-Ringer Buffer (KRB), and loaded with Fluo-3 Ca²⁺-indicator (4 µM, Invitrogen) for 30 min at room temperature, followed by washing and incubation at 37°C for 20 min. In some experiments 2 µM thapsigagrin, the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), in 0.5 mM EGTA, was used during the loading. Cover-slips were then transferred on imaging-chambers in KRB and analyzed on Leica TCS SP5 using a Leica HCX APO L water immersion objective. After initial recording of immature DCs, GNPs (10 µg/ml) and/or LPS were added. The light scattered from GNPs was detected upon excitation at 633 nm by a 660/30 nm Leica HyD hybrid detector (Leica Microsystems GmbH, Wetzlar, Germany). The images were acquired at a frequency of 2 Hz per channel for a total of 5 min. Similar analysis was performed on cells cultivated with LPS (100 ng/ml) and/or GNPs (10 µg/ml) for 24 h and 48 h. The fluorescence signals were expressed as ΔFt/F₀ ratios, ΔFt representing the fluorescence signal recorded at individual time points, minus F₀, the initial level of fluorescence. Areas under peaks and frequencies were calculated using Graph Pad Prism software (La Jolla, CA, USA).

Zebrafish (Danio rerio, strain AB/TL) were maintained and handled according to the guidelines from the Zebrafish Model Organism Database (http://zfin.org) and in compliance with the directives of the local animal welfare committee of Leiden University. Fertilization was performed by natural spawning at the beginning of the light period, and eggs were raised at 28.5°C in egg water (60 µg mL⁻¹ Instant Ocean sea salts). Mingle‐ or single‐lipoplex formulations (0.2 ng mRNA in 2 nL for each embryo) were injected into the hindbrain ventricle of 48 h post fertilization (hpf) zebrafish embryos. Embryos were anesthetized in 0.01% tricaine and embedded in 0.4% agarose containing tricaine before injection. Then, embryos were removed from the agarose. At indicated time‐points after injection, embryos were embedded again and imaged using confocal microscopy. Confocal z‐stacks were captured on a Leica TCS SPE confocal microscope, using a 10 × air objective (HCX PL FLUOTAR) or a 40 × water‐immersion objective (HCX APO L). Laser intensity, gain, and offset settings were identical between stacks and sessions. Images were processed by using the Fiji distribution of Image J.

For imaging experiments, we transferred individual slices into a bath chamber (Luigs & Neumann, Ratingen, Germany) at 37°C which was continuously superfused with bubbled (5% CO2, 95% O2) ECS. Imaging was performed on a Leica TCS SP5 AOBS Tandem II upright confocal system using a Leica HCX APO L water immersion objective (20 x, NA 1.0). Calbryte was excited with an argon 488 nm laser and the fluorescence detected by Leica HyD hybrid detector in the range of 500–700 nm (all from Leica Microsystems GmbH, Wetzlar, Germany). Images were acquired at a frequency of 10 Hz with 8-bit 256 X 256 pixels resolution at the tissue depth of around 15 μm to avoid the potentially damaged superficial cells. The thickness of the optical section was 4 μm to assure recording from a single cell layer. Before and after recording any time series, a high-resolution image (1024 X 1024 pixels) was taken for motion artefact assessment and region of interest (ROI) selection during analysis.