Fv3000 inverted lscm confocal system

MCF-7 cells were incubated with 10 μg/mL FITC-AAL, 10 μg/mL FITC-LTL, 50 nM Rhodamine-labeled TcsH, or 50 nM GFP-TcsH^1832–2618 in the medium on ice or at room temperature for 20 min. Cells were washed twice with ice-cold PBS, fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, stained with or without Hoechst, followed by fluorescence microscopy or flow cytometry analysis. Fluorescent images were captured using an Olympus FV3000 inverted LSCM Confocal System with the software FV31S-SW v2.3.2.169. For flow cytometry, cells were trypsinized, washed with PBS, resuspended in carbo-free blocking solution, and incubated for 30 min at room temperature. The cells were then washed and stained with 10 µg/mL FITC-LTL or 10 µg/mL FITC-AAL for 20 min at 4 °C, washed twice with cold blocking solution, and resuspended in PBS. Flow cytometry was carried out on a CytoFLEX LX (Beckman Coulter) and the data were analyzed with the software CytoExpert v2.4 (Beckman Coulter) and FlowJo v.10.8.0 (BD Biosciences).

MCF-7 cells were fixed with 4% PFA for 15 min at room temperature and then washed twice with PBS. Cells were blocked with 1% BSA in PBS for 30 min at room temperature but were not permeabilized. Cells were then incubated with Flag-tag antibody (1:200, conjugated with Alexa Fluor 488) for 1 h at room temperature. Cells were washed with PBS three times, followed by staining with Hoechst. Fluorescent images were captured using an Olympus FV3000 inverted LSCM Confocal System with the software FV31S-SW v2.3.2.169.