Tcs sp8 sted cw

MCF‐7 cells were seeded in a six‐well plate at a density of 3 × 10⁵ cells/well and cultured for 24 h at 37°C with 5% CO₂. Subsequently, a d‐pen solution (666 μM), a suspension of NH₂‐MIL‐101(Fe) (900 μg/ml), or a suspension of NH₂‐MIL‐101(Fe)/d‐pen (1000 μg/ml) containing an equivalent amount of d‐pen and NH₂‐MIL‐101(Fe) was added to each well and incubated for 6 h. Afterward, the cells were washed with fresh medium and incubated with 2 ml of 10 μM of DCFH‐DA for 30 min. DCFH‐DA was employed as a fluorescent probe for ^•OH detection, as it produced fluorescent 2′‐7′‐dichlorofluorescein (DCF) when reacted with ^•OH.⁴³ Thus, the treated cells were examined under a confocal microscope (CLSM; TCS SP8 STED CW, Leica Microsystems, Wetzlar, Germany) at excitation and emission wavelengths of 488 and 530 nm, respectively. To examine the effect of concentration, the experiments described above were repeated with a suspension of 500 μg/ml NH₂‐MIL‐101(Fe)/d‐pen, containing an equivalent amount of 333 μM d‐pen.

FRAP was performed on a laser scanning confocal microscope (TCS SP8 STED CW; Leica Microsystems) to i) assess the mobility of ligands in lipid bilayers and ii) to visualise the recovery of TIGIT and CD155 molecules within clusters. Mobility of ligands in PLBs or BSLBs was assessed using Streptavidin or ligand directly conjugated to Alexa-Fluor 647. Briefly, 1 μg/mL of either protein was loaded into bilayers, washed as described above and imaged using the 100× oil objective (NA, 1.4). 5 μm × 5 μm regions were bleached with 100% 660 nm STED laser and imaged every second for 60–90 s with the WLL set at 650 nm. Mobility could then be assessed by plotting the recovery within the bleached region over time, compared to adjacent 5 μm × 5 μm regions that had not been bleached. We routinely observe 100% recovery with T_1/2 ~ 5 s. To assess TIGIT and CD155 clusters in cells on PLBs, the same setup was used, except that TIGIT-GFP was imaged and bleached using the WLL at 488 nm, and CD155-AF647 imaged and bleached with the WLL at 650 nm, in defined ROIs that covered the entirety of a visible cluster. Recovery was then assessed by normalising recovery to clusters of similar intensity that were not bleached.