Tcs sp5x

For fluorescence microscopy, about 10 000 cells
in 200 μL of medium supplemented with 10% fetal bovine serum
and 1% penicillin/streptomycin were seeded in each well of an 8-well
chamber (μ-Slide 8 well; Ibidi) and cultured for 1–2
days. The medium was replaced with 200 μL of phenol red- and
serum-free RPMI-1640 or DMEM containing 10 μM glycosidase-reactive
fluorescent probe. The plate was incubated at 37 °C for 1 h in
a humidified incubator under 5% CO₂ in air, and differential
interference contrast (DIC) and fluorescence images were obtained
using a Leica Application Suite Advanced Fluorescence with a TCS SP5
X. The light source was a white light laser. Excitation and emission
wavelengths were 498 nm/500–600 nm.

Thrombus models were established
by following the published protocols.^{63 (link)} Fibrinogen from human plasma (1 mg/mL) as the
physiological source of thrombus was dissolved in a solution of 50
mM Tris-HCl (pH 7.4) and 140 mM NaCl. Thrombus formation was induced
by adding 1 U/mL thrombin and 2.5 mM CaCl₂ to the fibrinogen
solution, which was then cultured at 37 °C for 1 h. A turbidity
assay was used to monitor the process of thrombus formation by measuring
the absorbance (340 nm) at 10 min intervals. Fluorescent-labeled thrombus
was formed by an additional supply of 10 μL of Alexa Fluor 488-conjugated
fibrinogen to the fibrinogen solution containing thrombin and CaCl₂, followed by incubation at 50 °C for 2 h. The fluorescent
thrombus was characterized by CLSM (Leica, TCS SP5X). Thrombus models
and EM-JPMs (50 μL, ∼4.9 × 10⁵ per μL)
in a μ-slide 8 well (ibidi, Germany) were irradiated with a
760 nm laser at a laser power of 2.43 J/cm² (two-photon
laser, Chameleon Vision, Coherent, USA). Thrombus lysis was evaluated
by a fluorescence-based assay.