Cy3 nhs ester

A 1-μl aliquot of Cy3-NHS ester (Lumiprobe) dissolved in dimethyl sulfoxide (5 μg/ml) was mixed with 100 μl of the L. biflexa culture at room temperature. Excess dyes free from cells were removed by centrifugation at 1000g for 4 min and then suspended into the motility buffer. The cells labeled with the dyes were observed with a fluorescent microscope (BX53, UPlan-FLN 100×, U-FGW, Olympus), and their fluorescent images were acquired with a CCD camera (WAT-910HX/RC, Watec) at a frame rate of 30 Hz.

TKIs-loaded FRTs were labelled with Cy3 NHS ester (Lumiprobe, Hannover, Germany). Cells were seeded to chamber slide (30,000 cells per well) and incubated overnight. Then, the cells were treated with Cy3 NHS ester-labelled TKIs-loaded FRTs (normalized to 4 μM concentration of TKIs). After washing with PBS, cells were fixed using 200 μL of 4% formaldehyde in PBS and incubated for 10 min at 25°C. The permeabilization of membranes was achieved by 100 μL of 0.1% Triton X-100 in PBS. After blocking with 100 μL of 3% BSA in PBS, the chamber slides were incubated with anti-Rab5 (PA3-915, dilution 1:250; Thermo Fisher Scientific), anti-FR (ab3361, dilution 1:100; Abcam, Cambridge, UK) or anti-LAMP1 (14-1079-80, dilution 1:100; Thermo Fisher Scientific) primary antibodies and incubated overnight at 4°C. After washing, the relevant FITC-labelled secondary antibodies were added and the slides were incubated at 4°C, 1 h. After washing with PBS-T (3×), each well was stained with Hoechst 33342. Stained slides were visualized using CLSM (LSM 880, Carl Zeiss, Jena, Germany)

For fluorescent labeling of PF201, PF201 was dissolved in 0.1 M NaHCO₃ and then mixed with 100 mg/mL solution of Cyanine3 NHS ester (Cy3-NHS ester; Lumiprobe) at a ratio of 9:1. Following 4-h reaction under dark conditions, the Cy3-labeled PF201 solution was desalted using a Sephadex G-10 gel filtration column (GE Healthcare). The Cy3-labeled PF201 (100 mg/kg) was orally administered to 7-week-old male ddy mice (Shimizu Laboratory Supplies Co., Ltd.), and the stomach, small intestine, portal vein, and liver were collected at 1 h after the administration. The organs were homogenized in phosphate buffered saline (PBS). Extracts obtained from each organ were analyzed by HPLC (Nexera XR; Shimadzu) to detect Cy3-labeled peptides.