Fig. 1.

Antagomirs efficiently enter primary lymphocytes, are localized in the cytoplasm and show low cytotoxicity. Ex vivo isolated (MACS Technology, Miltenyi Biotech, Germany) unstimulated CD4⁺ T cells and unstimulated CD19⁺ B cells were treated with fluorescein-coupled non-targeting control Antagomir (Antagomir-scr) or incubated in serum-free medium without addition of Antagomir (Incubation ctrl). a) Cells were treated with concentrations of 0.125 to 2 μM of fluorescein-coupled Antagomir-scr for 2 h in serum-free medium and fluorescence was analyzed by flow cytometry; b) cells were treated with a concentration of 1 μM of fluorescein-coupled Antagomir-scr, washed after 2 h of incubation with PBS, cultured in FCS-containing RPMI medium for 3 days and fluorescence was analyzed by flow cytometry, d0 = cells after 2 h of incubation; c) cytoplasmic localization of fluorescein-coupled Antagomir-scr in live murine CD4⁺ T cells, murine CD19⁺ B cells, human CD4⁺ T cells and human CD19⁺ B cells was determined by single cell immunofluorescence analysis (Amnis ImageStreamX MKII, Merck Millipore) 24 h after incubation with Antagomirs, BF — bright field, CD4/CD19 — surface staining; d) viability of murine CD4⁺ Th1 cells and CD19⁺ B cells after treatment with different concentrations of Antagomir-scr in serum-free medium for 2 h. Viability was determined by propidium iodide staining, analyzed by flow cytometry and normalized to cells that were incubated in serum-free medium without addition of Antagomir.