Nis elements ar microscope imaging software

HeLa cells were cultured on 35-mm glass-bottomed dishes (#D35-20-0-TOP, Cellvis) at 37°C with 5% CO₂. The cells were then transfected with the indicated plasmids by using the Lipofectamine 3000 (Invitrogen) transfection kit according to the manufacturer’s instructions. After transfection for 24 h, samples were mounted on a Nikon Ti2 Inverted microscope equipped with a Yokogawa W-1 dual spinning disk scanhead and Micro-Scanner for photo-stimulation and a stage top incubator for live cell imaging. The captured images were analyzed by the NIS-Elements AR microscope imaging software (Nikon NIS-element AR version 4.0).

HCAEC were seeded on fibronectin-coated coverslips and irradiated when confluent with the stated doses. Seven days post-irradiation the adherent cells were rinsed twice with Hanks’ Balanced Salt Solution (HBSS) with added calcium and magnesium to preserve cell junctions (Thermo Fisher Scientific, cat. 14025050) and fixed in formalin for 10 min. Then cells were permeabilised with 0.1% Trition X-100 in HBSS for 10 minutes followed by, blocking in 2% Foetal Calf Serum in HBSS three times, 10 min each. Coverslips were then incubated with primary antibodies for VE-Cadherin and CLDN5 for 1 h at room temperature. Next, coverslips were subjected to three 10 min washes with 2% Foetal Calf Serum in HBSS and incubated with Alexa conjugated secondary antibodies for 1 h at room temperature. Coverslips were then washed again three times, 10 mins each in FCS/HBSS and mounted on slides with Vectashield with DAPI (Vector Laboratories, cat. H-1500). Imaging was performed by using Nikon Eclipse Ti inverted microscope, data analysis was performed with NIS- Elements AR Microscope Imaging Software (Nikon UK). The primary antibodies used for immunofluorescence experiments are listed in Table 2.

Stable transfected K562 cell lines were imaged at 37°C on a Nikon Ti-E microscope equipped with an incubation chamber (Okolab) using a 60 × oil immersion objective (NA 1.4, Nikon). A focused 488 nm laser was used for GFP excitation. Emission was measured between 500-550 nm. For translocation experiments all cell lines expressing GFP-HO-1 were imaged before and after incubation under hypoxia (1 % O₂) for 48 h. For cell viability experiments selected cell lines were incubated with 10 μM imatinib for 24 h, imaged and compared to cells without imatinib stimulation. For data collection and picture editing we used NIS-Elements Ar Microscope Imaging Software (Nikon Instruments Europe BV, Amsterdam, Netherlands).

For analysis of centrioles, the EpCAM‐captured cells were cytospun (1000g for 5 min) onto number 1.5, 13mm coverslips pretreated with poly‐L‐lysine (1mg·mL⁻¹ poly‐L‐lysine, Sigma Aldrich, St. Louis, MO, USA) for 1 h, washed 5 times with deionized water, and allowed to dry. Cells were then fixed with 100% ice‐cold methanol for 15 min. Fixed cells were blocked for 30 min in 3% BSA and 0.1% Triton X‐100 in PBS (PBSTx + BSA). Primary antibodies were incubated in PBSTx + BSA for 1 h at RT and washed three times in PBSTx, followed by secondary antibody incubation in PBSTx + BSA for 30 min at RT and two washes with PBSTx. Cells were counterstained with DAPI and mounted on glass slides with Prolong Gold antifade medium (Invitrogen). Cells were stained for centrin (Millipore, 04‐1624), pan‐cytokeratin (Abcam, ab7753), and exclusion markers (see above). Pictures were taken using the nis‐elements ar microscope imaging Software (Nikon, Melville, NY, USA) in Nikon Eclipse Ti‐E with ORCA‐Flash 4.0 Digital CMOS camera (Hamamatsu) florescent microscope with 10‐20X objectives in all the channels. Centrin foci were manually counted under 100X magnification and scored in CTCs and PBMCs.

Fluorescence imaging for optogenetically engineered tools was performed on a Nikon Eclipse Ti-E microscope equipped with an A1R-A1 confocal module with LU-N4 laser sources (argon-ion: 405 and 488 nm; diode: 561 nm). 60× oil or 40× oil objectives were used for high resolution imaging and Ca²⁺ imaging. A built-in 488-nm laser source (1% input) was used as a blur light source. For analysis of Ca²⁺ influx in HeLa cells, the green Ca²⁺ indicator GCaMP6s and mCh-tagged STIM1ct fragment were co-expressed. Both 488-nm and 561-nm laser sources were used to excite GFP and mCherry, respectively, with an interval of 8 s. The collected images were analyzed by the NIS-Elements AR microscope imaging software (Nikon, NIS-element AR version 4.0). 30–60 cells were selected to define regions of interest (ROI) for analyzing time-lapse images of Ca²⁺ influx. Photostimulation was applied using a built-in 488-nm laser source (1% input). All experiments were repeated three times. Data are shown as mean ± SEM.