Figure 2.
Human ILC2 cells enriched via the RosetteSep ILC2 kit and expanded in α-MEM readily proliferate ex vivo. Enriched human ILC2 cells were cultured in α-MEM (A-B) or IMEM (C-D) media with 20% FBS, and supplemented with 20 IU/mL IL-2, 50 g/mL IL-25 and IL-33 for 10 days. While ILC2 cells are homogeneous by day 10 of expansion, for the subset of experiments where a greater number of cells was needed for downstream analyses, cultures were maintained for an additional 4 to 11 days (total 14-21 days). (A) Flow cytometric profiles of 10 days cultured human ILC2 cells. Cells were stimulated by cell stimulation cocktail with protein inhibitors followed by intracellular staining. (B) A graph showing cell numbers of IL-13-producing ILC2 cells after 10 days of culture. Numbers in red denote fold change of ILC2 cell expansion. (C) Flow cytometric profiles of 10 day cultured ILC2 cells. Cells were stimulated by cell stimulation cocktail with protein inhibitors, and followed by intracellular staining. (D) Fold expansion of IL-13 producing ILC2 cells after 10 days in culture. Numbers in red denote fold change of ILC2 cells recovered on day 10. (E) Fold expansion of ILC2 cells using the RosetteSep kit over 13 days in culture. (F) Contour plot of expression of CD161 and CD294 on expanded ILC2 cells on day 10. (G) Histogram of GATA3 expression by intracellular staining. Gray filled area represents control cell negative expression. (H) Contour plot of expression of amphiregulin in expanded ILC2 cells on day 14. One representative of 2 experiments. SSC-A, side scatter area.

Human ILC2 cells enriched via the RosetteSep ILC2 kit and expanded in α-MEM readily proliferate ex vivo. Enriched human ILC2 cells were cultured in α-MEM (A-B) or IMEM (C-D) media with 20% FBS, and supplemented with 20 IU/mL IL-2, 50 g/mL IL-25 and IL-33 for 10 days. While ILC2 cells are homogeneous by day 10 of expansion, for the subset of experiments where a greater number of cells was needed for downstream analyses, cultures were maintained for an additional 4 to 11 days (total 14-21 days). (A) Flow cytometric profiles of 10 days cultured human ILC2 cells. Cells were stimulated by cell stimulation cocktail with protein inhibitors followed by intracellular staining. (B) A graph showing cell numbers of IL-13-producing ILC2 cells after 10 days of culture. Numbers in red denote fold change of ILC2 cell expansion. (C) Flow cytometric profiles of 10 day cultured ILC2 cells. Cells were stimulated by cell stimulation cocktail with protein inhibitors, and followed by intracellular staining. (D) Fold expansion of IL-13 producing ILC2 cells after 10 days in culture. Numbers in red denote fold change of ILC2 cells recovered on day 10. (E) Fold expansion of ILC2 cells using the RosetteSep kit over 13 days in culture. (F) Contour plot of expression of CD161 and CD294 on expanded ILC2 cells on day 10. (G) Histogram of GATA3 expression by intracellular staining. Gray filled area represents control cell negative expression. (H) Contour plot of expression of amphiregulin in expanded ILC2 cells on day 14. One representative of 2 experiments. SSC-A, side scatter area.

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