Vybrant™ DiI Cell-Labeling Solution

This is expected. DiD (which is far-red fluorescent) is never as uniform as DiI (which is orange fluorescent). If uniformity is desired, try increasing the label time and concentration, but it still isn't likely to be as uniform as DiI. CellMask Deep Red Plasma Membrane stain is much more uniform and is about the same wavelength as DiD. However, if you intend to do cell tracking over days, CellMask stain has not been tried for that application.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

A typical method is to label one cell line with orange fluorescent DiI C₁₈ and the other cell line with green fluorescent DiO C₁₈. These orange and green lipophilic cyanine dyes will stain the membranes of cells. Cells that fuse will then have both dyes, yielding a yellow color (when images are overlaid or cells are imaged in a dual-bandpass filter). These live cells can then be labeled with Hoechst 33342 (a cell-permeant blue DNA stain comparable in wavelength to DAPI), but only as an endpoint just before imaging (since DNA stains can interrupt DNA function).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Lipophilic cyanine dyes, such as DiI (Cat. No. D282), DiO (Cat. No. D275), DiD (Cat. No. D7757) or DiR (Cat. No. D12731), are commonly used. The longer the alkyl chain on the dye, the better the retention in lipophilic environments.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Factors to consider are size of tracer, method of delivery (injection, direct application to tissue, etc.), and if the tracer needs to be fixable. Here are some links to details about the various classes of neuronal tracers we offer and how to choose between them:

Neuronal Tracing (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing.html)
Choosing a Tracer (https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/fluorescent-tracers-of-cell-morphology-and-fluid-flow/choosing-a-tracer.html)
Imaging Analysis (http://assets.thermofisher.com/TFS-Assets/BID/Reference-Materials/bioprobes-50-journal.pdf)

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

This is not recommended. When these stains bind to DNA and RNA, they may affect the normal function of the nucleic acids, disrupting transcription, as well as replication. Other reagents, such as CellTracker dyes or Qtracker reagents are more optimized for tracking without disrupting normal activity. If a nuclear label is still desired, though, and the cells are mammalian and non-hematopoietic, CellLight nuclear reagents can transiently transfect cells to express GFP or RFP on a nuclear-expressing protein for up to several days without affecting function.

Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.