Zen 2.5d analysis tool

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).

The expression of the TIGAR protein and E6 viral oncoprotein within the HPV16-infected cervical cancer clinical samples by immunofluorescence staining, as well as the detection of cellular apoptosis using Annexin V-FITC/PI (or DAPI), were visualized confocal fluorescence-microscopy on a Zeiss LSM800 instrument using a Plan-Apochromat 20x/0.8 objective lens and ZEN OS software (Carl Zeiss Microscopy). The relative fluorescence-intensities of the Anti-TIGAR-specific (Rhodamine Red-X-positive) and Anti-HPV16 E6-specific (Alexa Fluor 488-positive) signals were graphically quantified using the Zen 2.5D analysis tool (Carl Zeiss Microscopy).