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Updated: Aug 7, 2026
Flow Cytometry-based Assay for the Monitoring of NK Cell Functions
Published on: October 30, 2016
Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human
John P Veluchamy1,2, María Delso-Vallejo3, Nina Kok1
1Glycostem Therapeutics, Oss, The Netherlands.
Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCRγδ, CD19, CD14, SYTOX® Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n = 6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n = 12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions.
Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCRγδ, CD19, CD14, SYTOX® Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n = 6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n = 12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions.
