Mlc 400 b laser box

Microscopy imaging was performed on a laser scanning confocal microscope (C1 si, Nikon) using a ×10 objective (CFI Plan Apochromat, Nikon) and a ×60 oil immersion objective (CFI Plan Apo VC, Nikon). The following lasers were used for excitation: a 405 nm continuous wave laser (Melles Griot 56ICS/S2695) for DAPI and Hoechst; a 488 nm continuous wave laser (Coherent Sapphire) for FD10, ATTO 488 and Alexa Fluor® 488; a 561 nm continuous wave laser (Melles Griot 85-YCA-010) for Alexa Fluor® 568 and a 640 nm continuous wave laser (Melles Griot 56ICS/S2695) for Alexa Fluor® 647. After photoporation, cells were incubated with 1 µg/mL Hoechst (Life Technology, Belgium) in CCM for 15 min at 37 °C. After the cells were washed twice with DPBS, time-lapse recordings were performed on a spinning disk confocal microscope (Nikon eclipse Ti-e inverted microscope, Nikon) equipped with an MLC 400 B laser box (Agilent technologies), a Yokogawa CSU-22 Spinning Disk scanner (Andor) and an iXon ultra EMCCD camera (Andor Technology, Belfast, UK). HeLa cells were imaged in a stage-top cell incubator (37°C with 5% CO₂ supplied, Tokai Hit) for 1 h with a time interval of 3 min using a ×60 oil immersion objective lens (CFI Plan Apo VC 60 × oil, Nikon, Japan).

Visualization and quantification of endosomal escape was performed based on the dequenching assay that was first published by Rehman et al. [30 (link)] Therefore, red-fluorescent oligonucleotides (AF647 ONs) were co-incorporated into the complexes. Cells were seeded in 96-well plates with glass bottom at a density of 10,000 cells per well and were allowed to attach overnight. Cell nuclei were stained with Hoechst 33342 staining (1 mg/mL in H₂O; 1000× diluted). Next, AF647 ON-containing complexes were added to the cells in Opti-MEM and incubated for 1 h at 37 °C. After washing off the complexes, the cells were provided with full cell culture medium and laser treatment was performed, as described above. After laser treatment, the cells were imaged using a spinning disk confocal (SDC) microscope (Nikon Eclipse Ti, Tokyo, Japan) equipped with an MLC 400 B laser box (Agilent Technologies, Santa Clara, CA, USA), a Yokogawa CSU-X confocal spinning disk device (Andor, Belfast, UK), an iXon ultra EMCCD camera (Andor Technology, Belfast, UK) and NIS Elements software (Nikon, Japan). A Plan Apo VC 60× 1.4 NA oil immersion objective lens (Nikon, City, Japan) was used to yield an image pixel size of 234 nm. Exposure time was set to 20 ms and the images were processed using ImageJ (FIJI).