DiR iodide (DiR iodide [1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide]) is an important near infrared (NIR) fluorescent dye, which has been widely used for in vivo monitoring of cells.

Stain adherent cells: Grow adherent cells on sterile glass coverslips. Remove coverslips from growth medium and gently drain off excess medium. Place coverslip in a humidity chamber. Pipet 100 μL of the dye working solution onto the corner of a coverslip and gently agitate until all cells are covered. Incubate the coverslip at 37℃ for 2-20 minutes. Drain off the dye working solution and wash the coverslips two to three times with pre-warmed growth medium. Microscopy Detection: DiI, 549/565 nm, XF32-Omega, 31002-Chroma; DiR, 748/780 nm, XF112-Omega, 41009-Chroma

Instructions:
I. Solution preparation
1. Mother solution preparation: DiR iodide is prepared with DMSO at a concentration of 1-5 mM.
2. Working solution preparation: Dilute the mother solution to a suitable buffer such as serum-free culture medium, HBSS or PBS to prepare a working solution of 1 to 5 μM.
II. Suspended cells
1. Centrifuge at 1000 g for 3-5 minutes at 4℃, discard the supernatant, and wash twice with PBS for 5 minutes each. The cell density is 1×10^6/mL.
2. Add 1 mL Di working solution and incubate at room temperature for 5-30 minutes.
3. Centrifuge at 400g for 3-4 minutes at 4℃ and discard the supernatant.
4. Wash twice with PBS for 5 minutes each.
5. Resuspend the cells with serum-free cell culture medium or PBS and observe with fluorescence microscopy or flow cytometry.
III. Adherent cells
1. Culture adherent cells on sterile coverslips.
2. Remove the coverslip from the culture medium and aspirate the excess culture medium.
3. Add 100 μL of working solution, shake gently to completely cover the cells, and incubate at room temperature for 5-30 minutes.
4. Wash twice with culture medium, 5 minutes each time. Observe by fluorescence microscopy or flow cytometry.