Aim image examiner software

U87MG-EGFP and CHO-EGFP cells were grown in collagen-coated 35 mm glass-base dishes (Asahi techno glass, Tokyo, Japan) for 24 h in growth medium. Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde at 4°C for 30 min. The cells were then incubated with the purified GCR or GC fusion proteins (2 μg) at 37°C for 1 h and washed two times with PBS. Confocal scanning laser microscopy was performed with a Zeiss LSM 510 instrument (Carl Zeiss, Oberkochen, Germany) with a 40x objective. The images were processed using Aim Image Examiner software (Carl Zeiss, Oberkochen, Germany).

For confocal microscopic analysis, NK92-MI and NKF cells were seeded at 2 × 10⁴ cells per chamber on Laboratory-Tek German borosilicate cover glass with eight chambers (Nunc, Rochester, NY, USA) and incubated for 24 h in growth medium. The cells were washed twice with 200 μL PBS. Subsequently, the cells were fixed and permeabilized with Fixation/Permeabilization buffer (BD Bioscience, San Jose, CA, USA) for 30 min at 4°C. The cells were then washed with 200 μL 1× BD wash buffer and incubated with phycoerythrin (PE)-conjugated anti-mouse CD90.1 (BD Bioscience) antibody at room temperature for 1 h followed by three washes with 200 μL 1× BD wash buffer. The slides were mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and covered with glass cover slips. Confocal scanning laser microscopy was performed using a Zeiss LSM 510 instrument (Carl Zeiss, Oberkochen, Germany) with a 40× oil objective, as indicated. The images were processed using Aim Image Examiner software (Carl Zeiss).