New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Accurate tracking of lymphocyte migration is crucial for understanding immune responses.
• In vitro labeling of lymphocytes with fluorochromes is a common method for in vivo studies.
• Selecting appropriate fluorochromes is essential for successful detection and analysis.

Purpose of the Study:

• To evaluate 16 fluorochromes for their ability to label viable lymphocytes in vitro.
• To determine the suitability of these fluorochromes for in vivo migration studies using fluorescence microscopy and flow cytometry.
• To identify optimal fluorochromes for both short-term and long-term lymphocyte migration tracking.

Main Methods:

• In vitro labeling of viable lymphocytes with 16 different fluorochromes.
• Assessment of fluorescence detectability using fluorescence microscopy and flow cytometry.
• In vivo migration studies with detection at various time points post-injection.

Main Results:

• Four intracellular dyes (H33342, BCECF-AM, Calcein-AM, CFSE) were suitable for in vivo studies.
• H33342, BCECF-AM, and Calcein-AM enabled short-term (2-3 days) migration studies.
• CFSE allowed for long-term tracking (up to 8 weeks) due to covalent coupling with macromolecules.
• Differential fluorescence profiles enabled short-term double labeling and subpopulation analysis.

Conclusions:

• Specific fluorochromes are effective for labeling lymphocytes for in vivo migration studies.
• CFSE is ideal for long-term lymphocyte migration studies.
• BCECF-AM and Calcein-AM are suitable for short-term studies and double labeling experiments.
• Fluorochromes with distinct spectral properties facilitate multicolor labeling for detailed immune cell tracking.