DiD，DiO，DiI，DiR and DiS  labeling membranes

Introdction

  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.

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

NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.

References

1.         Heinrich L, Freyria AM, Melin M, Tourneur Y, Maksoud R, Bernengo JC, Hartmann DJ. (2006) Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials. J Biomed Mater Res B Appl Biomater.

2.         Higashide T, Kawaguchi I, Ohkubo S, Takeda H, Sugiyama K. (2006) In vivo imaging and counting of rat retinal ganglion cells using a scanning laser ophthalmoscope. Invest Ophthalmol Vis Sci, 47, 2943.

3.         Kalchenko V, Shivtiel S, Malina V, Lapid K, Haramati S, Lapidot T, Brill A, Harmelin A. (2006) Use of lipophilic near-infrared dye in whole-body optical imaging of hematopoietic cell homing. J Biomed Opt, 11, 050507.

4.         Wang G, Anrather J, Glass MJ, Tarsitano MJ, Zhou P, Frys KA, Pickel VM, Iadecola C. (2006) Nox2, Ca2+, and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius. Hypertension, 48, 482.

5.         Wertz A, Rossler W, Obermayer M, Bickmeyer U. (2006) Functional neuroanatomy of the rhinophore of Aplysia punctata. Front Zool, 3, 6.

6.         Harrison TA, Perry KM, Hoover DB. (2005) Regional cardiac ganglia projections in the guinea pig heart studied by postmortem DiI tracing. Anat Rec A Discov Mol Cell Evol Biol, 285, 758.

7.         Huesa G, Anadon R, Yanez J. (2003) Afferent and efferent connections of the cerebellum of the chondrostean Acipenser baeri: a carbocyanine dye (DiI) tracing study. J Comp Neurol, 460, 327.

8.         Kiyohara Y, Endo K, Ide C, Mizoguchi A. (2003) A novel morphological technique to investigate a single climbing fibre synaptogenesis with a Purkinje cell in the developing mouse cerebellum: DiI injection into the inferior cerebellar peduncle. J Electron Microsc (Tokyo), 52, 327.

9.         Levai O, Strotmann J. (2003) Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice. Histochem Cell Biol, 120, 483.

10. Kalchenko et al., Use of lipophilicnear-infrared dye in whole-body optical imaging of hematopoietic cell homing. Journal of Biomedical optics, September/October 2006, Vol 11(5).

Figure 1. T-cells isolated from the spleen were fluorescently stained with DiR and i.v. injected (5x106 cells/mouse) into a Nu/Nu mouse. Images above taken 24hrs post injection with IVIS Spectrum  show cells homing to the spleen.

embryonic stem cells, bone marrow derived stem cells, adipose derived stem cells, lymphocytes and erythrocytes),  DiR stock was prepared by dissolving 25 mg in 3 mL ethanol. Working solution of 320 µg/mL was prepared by diluting 199 µL of stock solution in 5 mL PBS. T-cells isolated from the spleen were incubated with 320 µg/mL DiR. After 30 min incubation, cells were spun down for 3 min at 1000 rpm at 4 ºC resulting in a blue pellet. Cells were washed twice in PBS and injected intravenously (5 x 106 cells/mouse). Control group was injected with 5 x 106 cells/mouse in PBS. Mice were imaged with IVIS Spectrum  at 10 min, 1hr, 6hr and 24 hrs post injection. Ideal filter set for DiR imaging is 710 nm excitation and 760 nm emission. Mice were imaged dorsally as well as ventrally at all time points. Brain, bones, spleen, liver, lungs and kidneys were harvested for ex vivo imaging 24 hrs post injection.

Non-invasive in vivo imaging showed the homing process of injected T cells to the liver and spleen in real time, which was confirmed by ex vivo imaging.

1. NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.