Isolation and enrichment of infiltrated NK cells from tumor spheroids of cervical carcinoma. (A) Work flow of the cell fractionation. 96 tumor spheroids grown from CFSE-labeled CaSki or SiHa cells (d0) were co-cultured with primary human NK cells at an effector-to-target (E:T) ratio of 3:1 for 24 h. Tumor spheroids were collected, pooled, and non-infiltrated NK cells and residual target cells in the periphery of the tumor spheroids (P fraction) were removed by washing. Solid spheroids containing infiltrated NK cells were disintegrated and subjected to CD45 MACS-bead isolation leading to a flow through fraction (F fraction) containing spheroid cells and an elution fraction (E fraction) containing the CD45+ NK cells. Cells from all fractions were quantified by flow cytometry after SytoxBlue staining for discrimination of viable and dead cells. Tumor spheroids incubated in the absence of NK cells served as cytotoxicity control (CO). (B/E) Quantification of infiltrated NK cells isolated from tumor spheroids derived from CaSki (B) and SiHa (E) cells. Data are shown as total number of CD45+ cells per sample. (C/D/F/G) Analysis of cell viability. Proportions of viable and dead NK cells (C/F) and target cells (D/G) within the fractions are shown. Viable cells are shown as solid bars, dead cells as open bars. Data are shown as mean ± SEM of three individual experiments (n = 3).