Lymphocyte blastoid transformation assay by cytofluorography

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Lymphocyte activation is crucial for immune responses.
• Quantifying lymphocyte transformation traditionally involves time-consuming methods.
• Accurate measurement of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis indicates cell proliferation.

Purpose of the Study:

• To develop a rapid, automated, micromethod for quantifying blastoid transformation.
• To assess lymphocyte ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) content changes post-stimulation.
• To compare this new method with established techniques for lymphocyte proliferation assessment.

Main Methods:

• Utilized a supravital stain, acridine orange, for quantifying cellular RNA and DNA.
• Employed a cytofluorometric method for analyzing lymphocyte populations.
• Required minimal sample volume (0.2 ml) and offered rapid results (every 5 minutes).

Main Results:

• The method accurately quantifies total cell counts, increased RNA (I-RNA), and increased DNA (I-DNA) lymphocyte populations.
• Results demonstrated strong correlation with established H3 uridine and C14 thymidine uptake methodologies.
• Phytohemagglutinin dose-response measurements showed good agreement between the new and traditional methods.
• Blast cell counts obtained via Wright-Giemsa staining closely matched the I-RNA population counts.

Conclusions:

• The developed automated micromethod provides a fast and accurate way to measure lymphocyte blastoid transformation.
• This technique offers a reliable alternative to conventional methods for assessing lymphocyte activation and proliferation.
• The cytofluorometric approach using acridine orange is effective for quantifying RNA and DNA dynamics in stimulated lymphocytes.