Deltavision omx

HSCs were incubated with fluorescently labeled rLZ-8 in a 6-well plate containing DMEM medium (10% fetal bovine serum) for 30 min. Then, the cells were counted and inoculated on to the dried and ultraviolet-treated coverslips coated with polyethyleneimine at a density of 0.5×10⁵/cm², followed by PBST (PBS with 0.1% Tween 20) washes. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with PBS containing 0.5% saponin for 10 min. Then, the non-specific antibody binding was blocked by incubation with PBST containing 1% BSA and 22.52 mg/ml glycine for 30 min. Next, the cells were incubated with primary antibodies G-CSFR, CSF1R, GM-CSFRα, and GM-CSFRβ (Abcam, Cambridge, UK), respectively, in a humid chamber at 4°C overnight, followed by fluorescent-labeled secondary antibody in 1% BSA for 1 h in the dark. Finally, after washing with PBS, the cells on the coverslip were sealed on the glass slide to avoid drying and preserved at -20°C in the dark. The samples were observed under the Structured Illumination Microscope (SIM, DeltaVision OMX, GE Healthcare, Uppsala, Sweden).

For SIM imaging, precision coverslips with 170 µm thickness were used. The imaging was performed at RT on a GE Deltavision OMX super-resolution upright microscope with a Plan-Apochromat N ×60/1.42 Oil immersion objective, equipped with 405, 488, 568, 640 nm diode lasers and sCMOS detector camera. Images were acquired using AquireSR 4.4 software, reconstructed, and aligned using SoftWoRx 7.0 software as described before^{37 (link)}. GE immersion oil calculator was used to calculate the refractive index of oil to be used while imaging images stained with two or more fluorophores. SIM images were constructed from average intensity projections of Z-stacks using Stacks/Z-project and analyzed using 3DimageJ plugins in imageJ 1.53c software. For the line-scan analyses, mCherry-Caldesmon doublets (with a distance of ~0.2 μm between the two peaks) were chosen and manually centered on the analyzed ROI. The fluorescence intensities of mCherry-Caldesmon and the corresponding second protein were then measured along the ROI.

To quantify the number of insulin granules, we fixed the cells in 4% paraformaldehyde for 15 min, washed them twice in PBS, and incubated them for 15 min with 5% donkey serum. Cells were incubated with anti-insulin mouse monoclonal antibody (ab6995, Abcam, 1:300 dilution) for 1 h at room temperature with constant shaking. Coverslips were then washed 3 times in PBS and incubated for 1 h in PBS with the Alexa-Fluor-488 secondary antibody (Thermo Scientific – Life Technologies) followed by 3 washes in PBS. Samples were mounted in the glycerol buffer (refractive index 1.42) containing antifade reagent. To study mitochondrial morphology, INS-1E cells were transfected with GFP targeted to the mitochondrial matrix, and the next day fixed and mounted as described above.
High-resolution fluorescence microscopy was carried out using a DeltaVision OMX™ with the Blaze SIM Module (GE Healthcare) equipped with a 60 × 1.42, PlanApo N, Oil Immersion objective, 405 nm, 488 nm and 568 nm laser lines, and the OMX Standard filter set drawer. Images were acquired in structured illumination mode using a Z-spacing of 125 nm and reconstructed using Softworx software (GE Healthcare, Seattle, WA, USA).

Cells were prepared for live imaging as described above, with the addition of CellMask Orange Plasma membrane Stain (ThermoFisher) according to manufacturer’s instructions after media replacement with 1X HBS. Cells were imaged on a DeltaVision OMX (GE) using 60X TIRF objective in an environmental chamber set to 37°C, 2% CO2 and 5% O2. Images were taken from randomly selected fields of view. Each dish was imaged for a maximum of 30 minutes to minimize stress on the cells. Three dishes from two independent experiments were used for each condition.

For live cell microscopy, 2 × 10⁴ sGFP sender and 2 × 10⁴ αGFP receiver cells were seeded into 35 mm FluoroDish Cell Culture Dishes (World Precision Instruments, FD35-100) and immediately imaged under a DeltaVision OMX (GE Healthcare) microscope with a 60x oil objective lens (Olympus) in a humidified chamber at 37°C with 5% CO₂. One image was taken per minute for 3 hr. Images were collected with a cooled back-thinned EM-CCD camera (Evolve; Photometrics).
For fixed cell microscopy, sender and receiver cells were seeded at the ratios indicated in Supplementary file 2 with a total number of 1 × 10⁵ cells onto Neuvitro-coated cover slips (Thermo Fisher Scientific, NC0301187) in a 12-well cell culture plate. 24 hr after co-culture, cells were fixed in 4% paraformaldehyde (PFA) PBS solution at room temperature for 10 min and washed with PBS and distilled water three times each, before mounting onto slides using 50% glycerol. Images were captured using a Leica DMI6000B inverted microscope with an 40x oil objective lens. For quantification, GFP-containing receiver cells were counted. Multiple coverslips were analyzed across independent experiments (n = 10).