Goat fitc conjugated anti human igg antibody

The binding of the ADC and trastuzumab to HER2 was assessed using a fluorescence-activated cell sorter (Quanta apparatus, Beckman Coulter). For each cell line, one million cells were pelleted, washed with PBS/1% BSA and incubated with 10 µg/mL of trastuzumab or of the ADC on ice for 1 h. Then, cells were washed with PBS/1% BSA and incubated with a goat FITC-conjugated anti-human IgG antibody (F9512, Sigma-Aldrich, 1:200 dilution) on ice for 1 h. The irrelevant 13R4 antibody targeting beta-galactosidase was used as a negative control and incubation of cells with the secondary antibody was only used for background measurements. The gating strategy that was used for FACS analyses is presented in Figure S6.

Internalization of the trastuzumab-DNA mimic ADC in breast and ovarian cancer cells was studied by immunofluorescence. Briefly, 3 × 10⁴ cells were seeded on coverslips in 24-well plates. Two days later, exponentially growing cells were incubated for 2 h without or with 15 µg/mL of traztuzumab alone or of the ADC, at 4 °C or at 37 °C. Then, supernatants were removed and cells were washed twice with PBS-Tween 1% and once with PBS. Cells were then fixed by a 40 min incubation in formalin (3.7% formaldehyde in PBS) and permeabilized with 0.5% Triton X-100/PBS for 15 min. The cells were washed twice with PBS and incubated in PBS/BSA (2%) for 40 min. They were further incubated with a goat FITC-conjugated anti-human IgG antibody (F9512, Sigma-Aldrich, 1:200 dilution) for 90 min, washed three times with PBS-Tween 1% and three times with PBS and mounted with Everbrithe ^® (Biotium, San Francisco, CA, USA) with DAPI, and were visualized using an epifluorescence Zeiss Imager 2 (Zeiss, Germany).