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Human lymphocyte culture and chromosome analysis
1Human Genetics Laboratories, Department of Genetics and Developmental Biology, and Department of Pathology and Laboratory Medicine, University of Connecticut Health Center, Farmington, CT 06030-6140, USA.
CSH Protocols
|March 2, 2011
Summary
Phytohaemagglutinin (PHA) stimulates human T-cells in vitro, enabling chromosome analysis from peripheral blood. This method provides reliable GTW banding for clinical and research purposes.
Area of Science:
- Cytogenetics
- Molecular Biology
- Hematology
Background:
- Phytohaemagglutinin (PHA), a lectin from red kidney beans, is a potent mitogen for human T-cells.
- Peripheral blood offers a convenient source for human cell-based studies.
- Standard chromosome preparation methods are essential for clinical diagnostics and research.
Purpose of the Study:
- To describe a reliable method for preparing human chromosomes from peripheral blood.
- To detail a GTW banding technique for chromosome analysis.
- To highlight the utility of PHA-stimulated lymphocytes for cytogenetic studies.
Main Methods:
- In vitro culture of human peripheral blood with Phytohaemagglutinin (PHA).
- Induction of mitotic cells and harvesting at peak mitotic index (64-72 hours).
- Chromosome preparation involving protease treatment (trypsin) and Giemsa staining with Wright stain (GTW banding).
Main Results:
- Successful induction of mitotic T-cells from peripheral blood using PHA.
- Generation of metaphase cells suitable for cytogenetic analysis.
- Consistent and reliable Giemsa-T-Wright (GTW) banding patterns were achieved.
Conclusions:
- PHA-stimulated peripheral blood lymphocyte culture is a convenient and effective method for human chromosome preparation.
- The described GTW banding technique is reliable and adaptable for various human tissues.
- This approach facilitates both clinical and research-based cytogenetic investigations.
