Plan apochromat 63x 1.40 oil diciii objective lens

5×10⁴ CD4⁺CD45RA⁺ T cells were placed into 96-well plates (Corning) coated with fibronectin (10 μg/ml, # 3420-001-01, Trevigen). After 30 min at 37°C, non-adherent cells were removed and adherent cells were fixed, permeabilized and stained with anti-cortactin antibody (#PA5-29799, ThermoFisher) and rhodamine phalloidin (#R415, ThermoFisher Scientific). Nuclei were visualized with DAPI. The LSM710 system (Carl Zeiss), Plan Apochromat 63x/1.40 Oil DICIII objective lens (Carl Zeiss) was used to acquire images. Podosome-like structures and membrane ruffles were quantified using F-actin-cortactin co-localization.

CD4⁺CD45RO⁻ T cells were stimulated and cultured for 96 h under standard condition before staining with BODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) (#D3922, ThermoFisher Scientific). Cells suspensions were incubated with 3 μM BODIPY for 1 h at 37 °C, washed with 1×PBS twice, and fixed with paraformaldehyde solution, 4% in PBS (19943 1 LT, Affymetrix) for 15 min at RT. Cells were cytospun onto slides and mounted using ProLong Diamond Antifade Mountant with DAPI (P36962, ThermoFisher Scientific). Sections were imaged with a LSM710 system (Carl Zeiss); an EC Plan-Neofluar 40x/1.30 Oil DICIII objective lens (Carl Zeiss) and a Plan Apochromat 63x/1.40 Oil DICIII objective lens (Carl Zeiss) were used to acquire images. The Bodipy intensity was quantified using the LSM710 co-localization function.