Plan fluor 10 0.30 objective lens

The Annexin V-FITC/PI-stained samples to quantify cellular apoptosis and cytotoxicity were visualized by confocal fluorescence-microscopy on a Zeiss LSM800 instrument equipped with an Airyscan detector and stage CO₂ incubator, using a Plan-Apochromat 20 × 0.8 objective lens and Zeiss ZEN system software (Carl Zeiss Microscopy). The formation of virological synapses and viral transmission (i.e., determined by quantifying the relative percentages of Anti-HTLV-1 gp21^Env-positive huPBMCs) between the mitomycin C-treated HTLV-1+ SLB1/pLenti-GFP lymphoblasts and cultured huPBMCs were visualized by immunofluorescence-confocal microscopy using a Plan-Apochromat 20 × 0.8 objective lens. The relative fluorescence-intensities of the DAPI, Anti-HTLV-1 gp21^Env-specific (rhodamine red-positive), and GFP signals were graphically quantified using the Zen 2.5D analysis tool (Carl Zeiss Microscopy). The GFP-expressing HTLV-1+ SLB1/pLenti-GFP T-cell clones were screened by confocal fluorescence-microscopy on a Nikon Eclipse TE2000-U inverted microscope and D-Eclipse confocal imaging system, equipped with 633 nm and 543 nm He/Ne and 488 nm Ar lasers, using a Plan Fluor 10 × 0.30 objective lens and DIC phase-contrast filter (Nikon Instruments).

For analysis of focal adhesion dynamics, 7 × 10⁴ cells were seeded on collagen-coated glass-bottom plates 48 h post transfection of a paxillin-GFP construct. Transfected cells were incubated in serum-free RPMI1640 medium for 16 h and stimulated with 10% FBS. Cells were imaged using an Eclipse Ti2 microscope (Nikon) equipped with a DS-Qi2 monochrome camera (Nikon). Images were captured every minute for 1 h using a Plan Fluor 10×/0.30 objective lens (Nikon). Focal adhesion assembly was measured using NIS-Elements AR 5.20.00 software.

Cells (1 × 10⁵) were seeded on 0.5 kPa PAGs or dishes in RPMI1640 medium supplemented with 10% FBS and incubated for 10 h. The cells were equilibrated in a cage incubator H301-K-frame (Okolab, Pozzuoli, Italy) for 1 h. Cells were imaged using an Eclipse Ti2 (Nikon, Tokyo, Japan) with DS-Qi2 (Nikon) monochrome camera. Images were captured every 15 min for 12 h using a Plan Fluor 10×/0.30 objective lens (Nikon). Cell movement was measured using the NIS-Elements AR 5.20.00 software.