Trypan blue exclusion assay by flow cytometry

Area of Science:

• Cell Biology
• Immunology
• Biotechnology

Background:

• Dye exclusion assays, like trypan blue (TB), are standard for determining cell viability.
• Traditional TB assays have limitations, including dye uptake by live cells and analyst variability.
• Flow cytometry offers high sensitivity for cell analysis.

Purpose of the Study:

• To develop an alternative cell viability assay combining TB exclusion and flow cytometry.
• To address limitations of conventional TB assays for improved reliability and speed.
• To validate the use of TB-protein complexes' fluorescence for cell viability detection.

Main Methods:

• Utilized flow cytometry to detect fluorescence emitted by TB-protein complexes (TB/bovine serum albumin, TB/cytoplasmic proteins) at 660 nm.
• Optimized TB concentration to 0.002% (w/v) for distinguishing live (unstained) from dead (fluorescent) cells.
• Assessed TB's compatibility with green fluorescence markers (anti-CD3/FITC) in human T-cells.

Main Results:

• TB-protein complexes showed stable fluorescence emission at 660 nm for 30 minutes.
• Optimized TB concentration effectively differentiated live and dead cells without quenching green fluorescence.
• High correlation observed between TB+ and propidium iodide+ cells, validating the assay.

Conclusions:

• A flow cytometry-based TB exclusion assay provides a quick and reliable method for cell viability analysis.
• This method overcomes limitations of traditional TB assays, enhancing accuracy and reducing subjectivity.
• The assay is compatible with multicolor flow cytometry, expanding its utility in cell research.