Lsm 700 meta confocal microscopy

MDA-MB-231, BT-549 or MDA-MB-231 EGFR-KO cells (1.0 × 10⁵ cells/well in 24-well), previously seeded on a coverslip for 24 h, were incubated with BODIPY@PNPs-CL4 or BODIPY@PNPs-SCR, diluted at 2 µM dye concentration in culture medium, from 30 up to 60 min. After three washes with Dulbecco’s phosphate-buffered saline (DPBS), cells were fixed with 4 % paraformaldehyde in DPBS for 20 min. Then, cells were incubated with Wheat Germ Agglutinin Alexa Fluor 647-conjugated (WGA-647, Invitrogen, Carlsbad, CA), for 20 min at RT and washed three times with DPBS. Finally, nuclei were stained with 1.5 µM 4′,6-Diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich) in DPBS for 5 min and coverslips were mounted with glycerol/DPBS. Samples were visualized by Zeiss LSM 700 META confocal microscopy equipped with a Plan-Apochromat 63x/1.4 Oil DIC objective.

To visualize Gint4.T on the surface of PDGFRβ-positive murine cells, 4 T1 and NIH3T3 (1.0 × 10⁵ cells/well in 24-well), previously seeded on a coverslip for 24 h, were incubated with FAM-labeled Gint4.T or FAM-labeled Scr (500 nM-final concentration in BB) for 10 min at RT. PDGFRβ-negative BT-474 cells were treated in the same condition and used as negative control. After three washes in Dulbecco’s phosphate-buffered saline (DPBS), cells were fixed with 4% paraformaldehyde in DPBS for 20 min, washed three times in DPBS and incubated with 1.5 μM 4′,6-Diamidino- 2-phenylindole (DAPI, D9542, Sigma-Aldrich). Finally, coverslips were mounted with glycerol/DPBS. The fluorescence images were taken under a Zeiss LSM 700 META confocal microscopy equipped with a Plan-Apochromat 63x/1.4 Oil DIC objective.

SK-BR-3 and MDA-MB-453 cells (10⁵ cells/well in 24-well), previously seeded on a coverslip for 24 h, were incubated for 1 h with 500 nM of 10_12 or 10_12-CL4 conjugates in BlockAid™ Blocking Solution (Life Technologies) at RT. Then, cells were washed three times in PBS and fixed in PBS/PFA 4% for 20 min. For the fluorescence visualization of mAbs and conjugates, cells were incubated with FITC-labeled anti-human IgG antibody (Fluorescein (FITC) AffiniPure Goat Anti-Human IgG (H + L) 1:300, Jackson ImmunoResearch Laboratories Inc., Madison, WI, USA) for 1 h at RT and then washed three times with PBS. Finally, cells were incubated with 1.5 μM 4′,6-Diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich) and mounted with glycerol/PBS. Samples were visualized by Zeiss LSM 700 META confocal microscopy equipped with a Plan-Apochromat 63×/1.4 Oil DIC objective.