GoKawiilLabelling the cellular structures with ECL probes
Preparation of Ru(bpy) 3 2+-N-hydroxysuccinimide-ester-labelled antibody
1 ml, 1 mg ml−1 goat anti-rabbit immunoglobulin G (IgG) antibody (Huabio, HA1002) was dialysed (Viskase, Membra-Cel MD44) overnight at 4 °C in 3 l of 0.01 M phosphate buffer saline (PBS, pH 7.6, Sigma) and adjusted to a concentration of 2 mg ml−1. Then 1 mg of Ru(bpy) 2 (mcbpy-O-Su-ester)(PF 6 ) 2 (Aladdin, R131404) was dissolved in 50 µl dimethyl sulfoxide and added to the purified goat anti-rabbit IgG antibody immediately. The reaction mixture was incubated at 25 °C for 1 h. Subsequently, the unreacted Ru(bpy) 2 (mcbpy-O-Su-ester)(PF 6 ) 2 was removed using dialysis (4 °C, in 3 l PBS for 6 h, with buffer replacement every 2 h). The obtained labelled antibody was diluted with PBS to a final concentration of 1 mg ml−1 for use.
Cell culture and immunolabelling
The HeLa cells, COS-7 cells and MCF-7 cells (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) were cultured at 37 °C with 5% CO 2 in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) mixed with 10% foetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin solution (Sangon Biotech). When reaching about 90% confluency, the cells were digested by 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA, Thermo Fisher Scientific) solution for 1 min and then transferred to sterilized indium tin oxide (ITO) coverslips. After 24 h, the transferred cells were rinsed with PBS three times and immediately fixed for 30 min at room temperature with 4% paraformaldehyde (PFA, Sangon Biotech). The excess PFA was removed by washing the samples with PBS. Then the cells were permeabilized and blocked with 1% Triton X-100 (Sigma-Aldrich) mixed with 5% bovine serum albumin (BSA, Sangon Biotech) in PBS for 1 h at room temperature. After washing, the cells were stained by rabbit anti-alpha tubulin antibody (Abcam, ab52866) with a dilution of 1:100 in 5% BSA/PBS blocking buffer overnight at 4 °C. For mitochondrial imaging, the cells were stained with rabbit anti-TOMM20 antibody (Abcam, ab186735). For CEA imaging, the cells were stained with rabbit anti-carcinoembryonic antigen CEA antibody (Abcam, ab133633). For integrin imaging, the cells were stained with rabbit anti-integrin alpha 5 antibody (Abcam, ab275977) under the same conditions. The cells were then washed three times with PBS and stained with 10 µg ml−1 Ru(bpy) 3 2+-labelled goat anti-rabbit IgG antibody at 37 °C for 2 h. Finally, the cells were washed three times and imaged. Cell lines were authenticated using short tandem repeat analysis by the supplier. All cell lines were routinely tested and confirmed to be negative for mycoplasma contamination.
Labelling the cellular structures with CL and BL probes
Construction of mammalian expression vectors
CL and BL labelling of the intracellular targets was achieved by using a genetically encoded BRET probe (GeNL), which used NanoLuc luciferase as the donor and mNeonGreen fluorescent protein as the acceptor in this study. The only difference is that CL was applied to fixed cells, whereas BL was applied to live cells. The GeNL system was constructed according to the reference method26. All targeted sequences used for cell transfection are listed in Supplementary Table 2.
Cell culture and transfection
HeLa, COS-7 and MCF-7 cells were transfected at approximately 70–90% confluence with 0.25 μg of plasmid DNA using Lipofectamine 3000 (Thermo Fisher Scientific). After 12 h, the medium was replaced with phenol red-free DMEM for subsequent live-cell BL imaging.
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