DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide]

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Staining of microtumors with DIR-RGD-NP. Fluorescence spectral images from dissected intestines and the attached mesentery. Images shown are from mCherry channel (red; left column) and DIR channel (green; middle column). The merged images (right column) demonstrate the best colocalization of mCherry and DIR signals (white arrow) in animals that received DIR-RGD-NP. A representative animal for each delivery system is shown; (n = 4 for soluble DIR and DIR-NP; n = 12 for DIR-RGD-NP). Source: <strong>Novel approach for the detection of intraperitoneal micrometastasis using an ovarian cancer mouse model</strong> by Alvero et al., <em>Scientific Reports</em>, Jan. 2017.
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Ex/Em (nm)754/778
CAS #100068-60-8
Storage Freeze (<-15 °C)
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Category Cell Imaging,
Biomolecule Labeling
DiI, DiO, DiD and DiR dyes are a family of lipophilic fluorescent stains for labeling membranes and other hydrophobic structures. The fluorescence of these environment-sensitive dyes is greatly enhanced when incorporated into membranes or bound to lipophilic biomolecules such as proteins although they are weakly fluorescent in water. They have high extinction coefficients, polarity-dependent fluorescence and short excited-state lifetimes. Once applied to cells, these dyes diffuse laterally within the cellular plasma membranes, resulting in even staining of the entire cell at their optimal concentrations. The distinct fluorescence colors of DiI (orange fluorescence), DiO (green fluorescence), DiD (red fluorescence) and DiR (deep red fluorescent) provide a convenient tool for multicolor imaging and flow cytometric analysis of live cells. DiO and DiI can be used with standard FITC and TRITC filters respectively. Among them DiD is well excited by the 633 nm He-Ne laser, and has much longer excitation and emission wavelengths than those of DiI, providing a valuable alternative for labeling cells and tissues that have significant intrinsic fluorescence. DiR might be useful for in vivo imaging or tracing due to the effective transmission of infrared light through cells and tissues and low level of autofluorescence in the infrared range.

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide] to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =

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This protocol only provides a guideline, and should be modified according to your specific needs.

1. Prepare DiO, DiI DiD, DiS or DiR membrane stain solutions:

1.1    Prepare DMSO or EtOH stock solutions: The stock solutions should be prepared in DMSO or EtOH at 1-5 mM.

Note: The unused portion of the stock solution should be stored at -20 oC. Avoid repeated freeze/thaw cycles.

1.2  Prepare working solutions: Dilute the stock solutions (from Step 1.1) into a suitable buffer such as serum-free culture medium, HBSS or PBS to make 1 to 5 µM working solutions.

Note: The final concentration of the working solution should be empirically determined for different cell types and/or experimental conditions. It is recommended to test at the concentrations that are at least over a tenfold range.


2. Stain the cells in suspension:

2.1    Suspend cells at a density of 1 × 106/mL in dye working solution (from Step 1.2).

2.2    Incubate at 37 °C for 2–20 minutes. The optimal incubation time varies depending on the cell type. Start by incubating for 20 minutes and subsequently optimize as necessary to obtain uniform labeling.

2.3    Centrifuge the labeled suspension tubes at 1000 to 1500 rpm for 5 minutes.

2.4    Remove the supernatant and gently resuspend the cells in pre-warmed (37 °C) growth medium.

2.5    Wash two more times as Steps 2.3 and 2.4.


3. Stain adherent cells:

3.1    Grow adherent cells on sterile glass coverslips.

3.2    Remove coverslips from growth medium and gently drain off excess medium. Place coverslip in a humidity chamber.

3.3    Pipet 100 μL of the dye working solution (from Step 1.2) onto the corner of a coverslip and gently agitate until all cells are covered.

3.4    Incubate the coverslip at 37 °C for 2–20 minutes. The optimal incubation time varies depending on the cell type. Start by incubating for 20 minutes and subsequently optimize as necessary to obtain uniform labeling.

3.5    Drain off the dye working solution and wash the coverslips two to three times with growth medium. For each wash cycle, cover the cells with pre-warmed growth medium, incubate for 5-10 minutes and then drain off the medium.


4. Microscopy Detection:

4.1    The selection of DiD, DiO, DiI, DiS and DiR’s filter sets is summarized in Table 1.

4.2    For simultaneous detection of multiple dyes, multiband filter sets are available as follows:

a)     DiI and DiO = Omega XF52, Chroma 51004

b)    DiI and DiD = Omega XF92, Chroma 51007

c)     DiI, DiO and DiD = Omega XF93, Chroma 61005


5. Flow Cytometry Detection:

Cells labeled with DiO, DiI, DiD, DiS and DiR can be analyzed using the conventional FL1, FL2, FL3 and FL4 flow cytometer detection channels, respectively.


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