Splenocytes harvested from either naïve donor mice or BMT recipients were analyzed pretransplant or on D10, respectively. unless otherwise specified. Splenic cell suspensions were stained for surface T-cell (CD4, CD8) and activation markers (CD25, CD28, CD62L, CD69) along with intracellular Tumor Necrosis Factor Alpha (TNFα) and Interferon Gamma (IFNγ) and then analyzed by flow cytometry using a four-color C6 FlowCytometer (Accuri, Ann Arbor, Michigan) as previously described.20 (link) To analyze Forkhead Box P3 (FoxP3) expression, splenocytes were stained with anti-CD4 and anti-CD25, fixed and permeabilized, and then stained with intracellular anti-FoxP3 (eBioscience, San Diego, California). All monoclonal antibodies were purchased from BD Biosciences Pharmingen (San Diego, California) or eBioscience. Irrelevant fluorochrome rat isotype immunoglobulins were used as negative controls. Data were analyzed using CFlow software (Accuri) as previously described.20 (link),25 (link)
Protocol detail
Flow Cytometric Analysis of T-cell Activation
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Cd25
Cd62l
Cell
Donor
Flow cytometry
Fluorochrome
Ifnγ
Immunoglobulins
Intracellular
Isotype
Mice
Monoclonal antibodies
Splenic
T cell
Tnfα
Corresponding Organization : University Hospitals Seidman Cancer Center
Other organizations : Case Western Reserve University, Chiang Mai University, Nationwide Children's Hospital, Yale University, Athersys (United States), Sidney Kimmel Comprehensive Cancer Center
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Variable analysis
independent variables
- Donor mice (naïve or bone marrow transplant recipients)
- T-cell surface markers (CD4, CD8, CD25, CD28, CD62L, CD69)
- Intracellular Tumor Necrosis Factor Alpha (TNFα) and Interferon Gamma (IFNγ)
- Forkhead Box P3 (FoxP3) expression
- Splenic cell suspensions were analyzed pretransplant or on D10 for naïve donor mice and bone marrow transplant recipients, respectively
- Irrelevant fluorochrome rat isotype immunoglobulins were used as negative controls
- Negative control: Irrelevant fluorochrome rat isotype immunoglobulins
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