研究目的
To describe a method for isolating human regulatory T lymphocytes (Tregs) using fluorescence-activated cell sorting (FACS) with pre-enrichment via magnetic immunoselection, aiming for high yield and purity.
研究成果
The protocol provides an effective method for isolating human Tregs with high purity and yield, suitable for research and potential clinical applications. It highlights the advantages of FACS over magnetic methods for specificity and the importance of pre-enrichment to reduce sort time. Future work should address cryopreservation effects and scalability for clinical use.
研究不足
The method may have limitations in discriminating intermediate antigen expression levels with magnetic isolation, potential issues with cell viability during cryopreservation, and the need for clinical grade reagents for therapeutic applications. Spectral overlap in FACS can limit the number of fluorochromes used.
1：Experimental Design and Method Selection:
The protocol involves isolating peripheral blood mononuclear cells (PBMCs) from human blood using centrifugation over lymphocyte separation medium and erythrocyte lysis, followed by magnetic immunoselection to enrich CD25+ cells, and finally FACS to sort CD4+CD25+CD127lo Tregs.
2：Sample Selection and Data Sources:
Human blood samples such as buffy coat, leukocyte cone, or heparinized blood are used.
3：List of Experimental Equipment and Materials:
Includes centrifuge, MACS separator, flow cytometer (e.g., BD FACSAria), antibodies (e.g., anti-CD4-ECD, anti-CD25-PE Cy7, anti-CD127-PE), and reagents like Lymphocyte Separation Medium and PharmLyse solution.
4：Experimental Procedures and Operational Workflow:
Steps include PBMC isolation, CD25+ enrichment with magnetic beads, immunostaining with fluorescent antibodies, and FACS sorting with specific gating strategies.
5：Data Analysis Methods:
Purity and yield are assessed using flow cytometry analysis post-sort.
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Lymphocyte Separation Medium
LSM
PAA
Used for isolating peripheral blood mononuclear cells (PBMCs) from human blood by centrifugation.
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PharmLyse solution
BD Pharmingen
Used for erythrocyte lysis in PBMC isolation.
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Human CD25 microbeads
Miltenyi Biotech
Used for magnetic immunoselection to enrich CD25+ cells.
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MACS separator
Miltenyi Biotech
Equipment for magnetic cell separation.
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MACS magnet
Miltenyi Biotech
Part of the MACS system for magnetic separation.
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MACS columns
LS or MS
Miltenyi Biotech
Columns used in magnetic separation for cell isolation.
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Anti-CD4-ECD antibody
clone SFCI12T4D11
Beckman Coulter
Fluorochrome-conjugated antibody for immunostaining CD4 antigen.
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Anti-human CD25-PE Cy7 antibody
clone M-A251
BD Biosciences
Fluorochrome-conjugated antibody for immunostaining CD25 antigen.
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Anti-human CD127-PE antibody
clone hIL-7R-M21
BD Biosciences
Fluorochrome-conjugated antibody for immunostaining CD127 antigen.
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Flow cytometer
BD FACSAria
BD Biosciences
Used for fluorescence-activated cell sorting (FACS) to isolate Treg cells.
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FACSFlow solution
BD Biosciences
Sheath fluid used in the flow cytometer during cell sorting.
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