Improved microplate fluorometer counting of viable tumor and normal cells

Area of Science:

• Biotechnology
• Cell Biology
• Analytical Chemistry

Background:

• Accurate cell counting is crucial for biological research and drug development.
• Existing methods like Coulter counters can be complex or require cell lysis.
• Fluorescence-based assays offer potential for sensitive, non-destructive cell enumeration.

Purpose of the Study:

• To develop and validate an improved, semiautomated method for in situ cell counting.
• To assess the sensitivity and reliability of the esterase activity-based fluorometer assay.
• To quantify and account for fluorescence variations affecting cell counting accuracy.

Main Methods:

• Utilized carboxyfluorescein diacetate to measure intracellular esterase activity as an indicator of viable cells.
• Developed a semiautomated microplate fluorometer assay for high-throughput analysis.
• Investigated sub- and supranormal fluorescence events using a menadione/U-87 MG cell model.

Main Results:

• The assay accurately estimated viable cell numbers ranging from 5 x 10(2) to 2.6 x 10(5) cells/well in a tumor cell line.
• Sensitivity down to 10(3) cells was demonstrated in two additional cell lines.
• Close correlation was observed between the fluorometer method and Coulter counter method after accounting for fluorescence anomalies.

Conclusions:

• The esterase activity-based fluorometer assay is a sensitive, reliable, and semiautomated method for viable cell counting.
• This technique offers a valuable alternative to traditional cell counting methods, particularly in microplate formats.
• Understanding and controlling fluorescence variations are key to ensuring accurate cell enumeration.