Na 1.5 oil objective

The μ-Slide 8 Well Glass Bottom chamber (ibidi) was coated with recombinant human ICAM-1-Fc (5 μg/mL) at room temperature for 3 h. Differentiated MFN2 KD and control HL60 cells (2 × 10⁶ cells/mL) cells were incubated with CellTracker Orange CMRA (4 μM) at room temperature for 1 h. After 2 washes with PBS, cells were incubated with unconjugated mouse anti-human CD18 blocking Ab (TS1/18, 4 μg/mL) or mouse IgG1κ isotype control (4 μg/mL) at room temperature for 10 min. After 2 washes with PBS, cells were resuspended in PBS plus 1 μM Mn²⁺, added into the chamber, and centrifuged at 500 × g at room temperature for 5 min to induce spreading. Cells were fixed with 1% PFA at room temperature for 5 min and washed twice with PBS to remove unadhered cells. Total internal reflection fluorescence (TIRF) images (Fig. 4A) were acquired with an iX83 Olympus inverted microscope equipped with a SAFe Light module (Abbelight, includes four color lasers, λ = 405 nm, 488 nm, 532 nm, and 640 nm), sCMOS fusion cameras (Hamamatsu), and a 100× NA 1.5 oil objective. A TIRF incidence angle of θ = 70° was used. The area of cell footprint was quantified by the “analyzing particles” function in FIJI-ImageJ2^{58 (link)} (Fig. 4B).

Differentiated MFN2 KD and control HL60 cells (5 × 10⁵ cells/mL) were incubated with fMLP (100 nM) or PMA (100 nM) or vehicle control at room temperature for 20 min. Cells were fixed with 1% PFA at room temperature for 10 min, washed twice with intracellular staining perm wash buffer plus 5% goat serum, and stained with AF568-conjugated phalloidin (5 U/mL) in intracellular staining perm wash buffer plus 5% goat serum at room temperature for 30 min. After 2 washes with PBS, cells were added into μ-Slide 8 Well Glass Bottom chamber (ibidi) pre-coated with 0.01% Poly-l-lysine (at 4°C overnight) and centrifuged at 500 × g at room temperature for 5 min to let the cells settle and adhere. Epifluorescence images (Fig. 3A)were acquired by using an iX83 Olympus inverted microscope equipped with the SAFe Light module (Abbelight, includes four color lasers, λ = 405 nm, 488 nm, 532 nm, and 640 nm), sCMOS fusion cameras (Hamamatsu), and a 100× NA 1.5 oil objective. The phalloidin median fluorescence intensity (MFI), which reflects the actin polymerization, was quantified by the “analyzing particles” function in FIJI-ImageJ2^{58 (link)} (Fig. 3B).