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Detection of unseparated human lymphocytes by flow cytometry
1Warren Grant Magnuson Clinical Center National Institutes of Health, Bethesda, Maryland, USA.
Current Protocols in Immunology
|April 25, 2008
Summary
This study presents a flow cytometry protocol for whole human peripheral blood, improving B cell recovery and reducing processing time. Careful lymphocyte gating is essential for accurate analysis of these key immune cells.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Flow cytometry is crucial for analyzing human peripheral blood cells.
- Traditional methods often involve Ficoll-Hypaque purification of mononuclear cells.
- Cell separation can lead to time delays, increased blood volume requirements, and potential cell loss.
Purpose of the Study:
- To develop and present a flow cytometry protocol for direct analysis of unseparated human peripheral blood cells.
- To optimize B cell recovery and reduce sample processing time.
- To highlight the importance of precise gating strategies for lymphocyte identification.
Main Methods:
- Analysis of unseparated, whole peripheral blood cells.
- Flow cytometry techniques applied directly to unprocessed blood samples.
- Comparison with methods using Ficoll-Hypaque-purified mononuclear cells.
Main Results:
- The whole blood method is faster and requires smaller blood volumes.
- Significantly greater B cell recovery is achieved with the whole blood approach.
- Lymphocyte gating requires careful attention due to their minority status in whole blood.
Conclusions:
- Direct flow cytometry analysis of whole peripheral blood offers advantages in time efficiency and B cell yield.
- This protocol is suitable for human peripheral blood studies.
- Accurate lymphocyte identification through meticulous gating is critical for reliable results.
