IR 700DX

Single-molecule imaging of cells^[3]
1. Near-infrared dyes and dye-protein conjugates
1) IRDye 700DX was conjugated to biotin-transferrin using an NHS-ester dye derivative.
2) Free dye was separated from the conjugate using a PD-10 desalting column.
2. Dye fixation
1) 35 mm glass bottomed dishes are cleaned with Piranha solution before being covered with 1 mg/ml BSA-biotin in Tris-EDTA (TE) buffer for 10 min at room temperature.
2) After washing with TE buffer, dishes are covered with 0.2 mg/ml streptavidin in TE buffer for 10 minutes.
3) Dishes are washed again in TE buffer before being incubated with a 0.05 nM solution of a single biotin-transferrin-NIR dye conjugate for 10 min at room temperature.
4) Excess dye conjugate is washed off with TE buffer and immobilised dye samples are stored at 4°C before imaging.
3. Cell sample preparation
1) MCF-7 cells are cultured on 35 mm glass bottomed dishes in RPMI 1640 media supplemented with glutamine and fetal calf serum, at 37°C in the presence of 5% CO₂.
2) Cells were incubated in media without serum for 18 hours before being labelled with 0.05 nM biotin-transferrin-IRDye 700DX (0.1 nM total biotin-transferrin concentration) for 1 h at 4°C.
3) Excess biotin-transferrin was washed off with cold PBS and cells were fixed with 4% paraformaldehyde. Samples were stored at 4°C until ready to image.
Single-molecule fluorescence imaging
1) Images of MCF-7 cells unlabeled or labeled with transferrin-IRDye 700DX were acquired at 10 Hz using epi or TIRF illumination.
2) Images of labeled cells were acquired while illuminating with 695 nm and 780 nm light simultaneously.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.