A1r a1 confocal laser microscope

LS174T cells were seeded onto an 8-well chambered coverglass (Thermo Scientific, Rockford, IL) at a density of 10⁵ cells/well and incubated in complete DMEM medium with 10% FBS. When the cells were bound to the bottom of the chambers, TRITC-labeled TAT-Gel was added to the wells either: 1) alone, 2) with heparin (TAT-Gel/Hep) or 3) with heparin and protamine (“TAT-Gel/Hep+Pro”), and incubated at 37°C for 3 h. The TAT-Gel/Hep complex was prepared by mixing TRITC-labeled TAT-Gel with 3-fold molar excess of heparin and incubation at 4°C for 30 min. The “TAT-Gel/Hep+Pro” treatment was by addition of protamine (3-fold molar excess to heparin) to the wells immediately after the cells were treated with TATGel/Hep. After incubation, cells were stringently washed for three times with heparin/PBS, followed by another wash with PBS, and the images of the live cells were acquired by a Nikon A1R-A1 confocal laser microscope with a 20×objective (Nikon Instruments Inc., Melville, NY) and analyzed using NIS-Elements Microscope Imaging software (Nikon Instruments Inc., Melville, NY).

LS174T and HCT116 cells were seeded on Nunc™ Lab-Tek™ Chambered Coverglass (Thermo Scientific) at a density of 10⁴ cells per well and incubated for overnight. After incubation, cells were treated with either TRITC-labeled gelonin, TRITC labeled-TAT-Gel or TRITC labeled-TAT-Gel/Dylight 488 labeled-T84.66-Hep complex (a.k.a. TAT-Gel/T84.66-Hep) and then incubated at 37°C for 3 h. Final concentrations of gelonin and TAT-Gel were 1 μM, and the TAT-Gel/T84.66-Hep was prepared by mixing the two components at 1:3 molar ratios (TAT : heparin). For a group of LS174T cells pre-treated with TAT-Gel/T84.66-Hep, protamine (3-fold molar excess of heparin) was added to the wells immediately following the complex treatment. After incubation, cells were washed with PBS and the nuclei of the cells were counterstained with Hoechst 33342. The images of live cells were acquired by using a Nikon A1R-A1 confocal laser microscope (Nikon Instruments Inc., Melville, NY) with a 60× objective and analyzed using NIS-Elements microscope imaging software (Nikon Instruments Inc., Melville, NY).

LS174T cells were seeded onto an 8-well chambered coverglass (Thermo Scientific, Rockford, IL) at 10⁵ cells/well and incubated in complete DMEM medium with 10% FBS. When the cells were attached to the bottom of the chambers, TRITC-labeled rGel and TAT-Gel were separately added to the wells and incubated at 37°C for 3 h in humidified CO₂ incubator. After incubation, the cells were rinsed three times with heparin/PBS (10 mg/mL heparin, 20 mM phosphate buffer, 0.15 M NaCl, pH 7.4) and their nuclei were counterstained with Hoechst 33342. Following the Hoechst staining, the cells were washed three times with PBS and the images of the live cells were taken by a Nikon A1R-A1 confocal laser microscope with a 60×objective (Nikon Instruments Inc., Melville, NY). Cell images were acquired and analyzed using NIS-Elements Microscope Imaging software (Nikon Instruments Inc., Melville, NY).