A trypsin-based method for isolating leukocytes from human choriodecidua suitable for immunophenotyping and...

Area of Science:

• Reproductive immunology
• Cellular and molecular biology

Background:

• Leukocytes at the maternal-fetal interface are crucial in labor inflammation.
• Current isolation methods (scraping, enzyme digestion) hinder downstream analyses like immunophenotyping and transcriptomics.

Purpose of the Study:

• To develop an effective leukocyte isolation method from the choriodecidua for advanced analyses.
• To ensure isolated leukocytes are suitable for flow cytometry and RNA-based techniques.

Main Methods:

• Gentle trypsin digestion of the choriodecidua.
• Isolation of leukocyte-enriched cell mixtures.
• Assessment of lymphocyte and myeloid cell viability.
• Evaluation of cell surface marker integrity for flow cytometry.
• RNA extraction for quantitative real-time PCR (qRT-PCR) and microarray analysis.

Main Results:

• The gentle trypsin digestion method yields a leukocyte-enriched cell population.
• High viability was observed for lymphocytes, with reduced viability in myeloid cells.
• Isolated leukocytes maintained cell surface marker integrity, suitable for flow cytometry.
• High-quality RNA was successfully obtained for qRT-PCR and microarray analyses.

Conclusions:

• Gentle trypsin digestion is a viable method for isolating leukocytes from the maternal-fetal interface.
• This method supports downstream immunophenotyping and transcriptomic analyses, crucial for studying labor mechanisms.
• The technique offers improved cell recovery and suitability for molecular analyses compared to traditional methods.