Dapi staining

The proportions of senescent cells in both hUVECs and hFBs were determined using the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, Missouri, USA). Cells were seeded in 6-well plates at a density of 2 × 10⁴ cells/cm² and incubated with FITC-SiO₂-COOH NPs at concentrations of 50 μg/mL or 100 μg/mL at 37 °C and 5% CO₂ for 48 h. Post incubation, the culture medium was aspirated and cells were briefly rinsed with phosphate-buffered saline (PBS) prior to fixation with 1X Fixation buffer for 7 min. Following two PBS washes, cells were treated with the staining mixture and incubated overnight at 37 °C in a 5% CO₂ environment. After discarding the staining mix and rinsing with PBS, cells underwent DAPI staining (Abcam, Cambridge, UK) for 15 min. The samples were visualized and documented using both optical and fluorescence microscopes (Olympus, Tokyo, Japan), with the resultant images analyzed using ImageJ software (version 1.46r).

The effect of the dietary intake of High OC/OL-EVOO on apoptosis was also investigated by TUNEL Assay in isolated peripheral blood mononuclear cells (PMBC) isolated from peripheral blood from the CLL patients during the dietary intervention. Approximately 20 ml of venous blood from the patients with CLL were obtained in a heparinized syringe (heparin, 90 IU/10 ml blood). The blood was overlaid on Ficoll–Paque (Histopaque 1077, Sigma, Aldrich Company Ltd, Dorset, UK) at a 2:1 ratio and centrifuged at 400g for 35 min at 20°C. PBMCs were collected from the interface and washed with PBS buffer (GIBCO Invitrogen Corporation, Carlsbad, CA, USA) three times to remove the plasma and the Ficoll, according to the manufacturer’s instructions. Isolated PBMC were transferred to the slides and fixed in 4% formaldehyde in PBS (pH 7.4) for 30 min at 4°C. Then the cells were washed twice in PBS and permeabilized in PBS containing 0.2% Triton x-100 for 30 min at room temperature. After washing with PBS, TUNEL was performed according to the manufacturer’s instructions (Biotium, Inc. Fremont, CA USA). DAPI staining at concentration 0.5 μM (Abcam UK) was performed incubating the slides for 30 min after the TUNEL assay.

The heart, liver, spleen, lungs, kidneys, and tumor masses were dissected. All dissected mice were sacrificed 39 days after subcutaneous tumor seeding. Tissue specimens were fixed with 4% buffered formaldehyde, and paraffin sections were analyzed using hematoxylin-eosin staining and immunohistochemistry. H&E staining of major organs was used for the preliminary safety evaluation of CAR-T cells in vivo, and immunohistochemical staining of the spleen was used to evaluate CAR-T cell persistence and infiltration. The primary and secondary antibodies used in this study were rabbit anti-human CD3 and hrp-labeled goat anti-rabbit IgG H&L (Abcam, Cambridgeshire, GB, UK), respectively, followed by DAPI staining (Abcam, Cambridgeshire, GB, UK) for nuclear staining.