Multicolor Flow Cytometry Evaluation of Lymphocyte Subpopulations

A multicolor flow-cytometry-based approach was used to assess frequency, phenotype and functionality of the major circulating lymphocyte subpopulations and NK cells, before (pre), during (T92) and after treatment (4 and 6 months after enrollment).
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient (Lymphoprep Axis-Shield, Scotland) and frozen as described elsewhere (29 (link)). Samples taken at different time points were tested within the same experimental session, in accordance to “minimal information about T cell (MIATA) and NK (MIANKA) assays” guidelines (http://miataproject.org/miata-guidelines/final-guidelines-2/), to improve the data quality level of flow cytometry assay. Cells were thawed in the presence of DNase. Live and dead cells were discriminated by trypan blue exclusion method and samples showing viability less than 70% were not further processed.

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Moschella F., Buccione C., Ruspantini I., Castiello L., Rozo Gonzalez A., Iacobone F., Ferraresi V., Palermo B., Nisticò P., Belardelli F., Proietti E., Macchia I, & Urbani F. (2023). Blood immune cells as potential biomarkers predicting relapse-free survival of stage III/IV resected melanoma patients treated with peptide-based vaccination and interferon-alpha. Frontiers in Oncology, 13, 1145667.

Publication 2023

Ficoll gradient

After treatment Assay Axis Cells Dnase Ficoll Flow cytometry Frozen Lymphoprep Nk cells Peripheral blood mononuclear cells Phenotype T cell Trypan blue

Corresponding Organization : Istituto Superiore di Sanità

Other organizations : Istituti di Ricovero e Cura a Carattere Scientifico, Istituto di Farmacologia Traslazionale, National Research Council, University of Rome Tor Vergata

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Variable analysis

independent variables

Treatment time points (pre, T92, 4 months, 6 months)

dependent variables

Frequency of major circulating lymphocyte subpopulations and NK cells
Phenotype of major circulating lymphocyte subpopulations and NK cells
Functionality of major circulating lymphocyte subpopulations and NK cells

control variables

Peripheral blood mononuclear cells (PBMCs) isolated by Ficoll gradient
Samples tested within the same experimental session
Cells thawed in the presence of DNase
Live and dead cells discriminated by trypan blue exclusion method
Samples with viability less than 70% not further processed

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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