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Updated: Aug 8, 2026
The Use of Flow Cytometry to Assess the State of Chromatin in T Cells
Published on: December 17, 2015
Simultaneous cytofluorometric analysis for the expression of cytoplasmic antigens and DNA content in CD3- human
M R Zocchi1, F Marelli, A Poggi
1Laboratorio Immunoterapia Adottiva, Istituto Scientifico San Raffaele, Milan, Italy.
Insights
This study introduces a two-color immunofluorescence method to analyze cytoplasmic antigens and cell cycle phases simultaneously. The method reveals intracellular CD3 and CD28 expression in thymocytes entering cell cycle, linking T cell marker appearance to thymocyte development.
Area of Science:
- Immunology
- Cell Biology
Background:
- T cell development involves complex antigen expression changes.
- Simultaneous analysis of cytoplasmic antigens and cell cycle status is challenging.
Purpose of the Study:
- To develop a novel two-color immunofluorescence staining method.
- To analyze cytoplasmic T cell antigens and cell cycle entry in thymocytes.
- To investigate the relationship between intracellular T cell markers and cell cycle progression.
Main Methods:
- Two-color immunofluorescence staining.
- Analysis of CD3- thymocytes from thymus cell suspensions.
- Cell sorting, fixation, permeabilization, and indirect immunofluorescence.
- Monoclonal antibody staining for T cell markers (CD3, CD2, CD28, TCR α/β, TCR γ/δ).
- DNA content analysis for cell cycle phase determination.
Main Results:
- Developed a method for simultaneous cytoplasmic antigen and cell cycle phase analysis.
- Detected intracellular CD3 and CD28 in >80% of thymocytes.
- Majority of S/G2/M phase thymocytes expressed intracellular CD3 and CD28.
- Intracellular CD3/CD28 expression correlated with cell cycle entry.
Conclusions:
- The novel method allows simultaneous detection of cytoplasmic antigens and cell cycle phases.
- Intracellular CD3 and CD28 expression is prevalent in thymocytes entering the cell cycle.
- Findings suggest a link between cytoplasmic T cell marker appearance and thymocyte development stages.
Abstract:
We describe a method of two-color immunofluorescence staining which allows the simultaneous analysis of both cytoplasmic antigens and cell entry into the S/G2/M cell cycle phases. This analysis was performed on CD3(-)-activated thymocytes obtained from either highly purified CD1-CD3-CD4-CD8- cells or fresh thymus cell suspensions, stimulated with low doses of phorbol-12 myristate-13 acetate (0.5 ng/ml) and interleukin-2. On the 14th day under these culture conditions about 90% of thymocytes did not express CD3 antigen on the cell surface. CD3- cells were further purified by cell sorting, fixed in paraformaldehyde, and permeabilized with Nonidet-P40. Then these thymocytes were stained by indirect immunofluorescence with monoclonal antibodies identifying T cell-specific molecules (CD3, CD2, CD28, TCR alpha/beta, and TCR gamma/delta) and analyzed for DNA content. Interestingly, both CD3 and CD28 antigens were detectable in the cytoplasm of most cells (greater than 80%). Further, the majority of the thymocytes which had entered the S/G2/M phases of the cell cycle (20%) expressed intracellular CD3 and CD28 molecules and reacted with the anti-beta framework beta F1 monoclonal antibody. The relationship between the appearance of CD3 and other T cell markers in the cytoplasm, the cell cycle entry, and the thymocyte development is discussed.
