Fig. 5.

Flow cytometric quantification of cellular association of nanoplexes in leaky gut model. Leaky gut model was treated with fluorescently-labeled nanocarrier (DiI-LNPs) and siRNA (AF647-siJAK1). Co-culture was incubated twice apically for 6 h with nanoplexes (N/P ratio of 8) at two different concentrations with LNPs at 45 μg or 90 μg per well and siJAK1 at 135 nM (Low) or 270 nM (High), respectively. Control treatments were co-cultures without nanocarrier and siRNA (Control), blank LNPs (90 μg/mL) or naked siJAK1 (Low/High). After co-culture dissociation, the resulting cell suspension was stained for living (Calcein AM; 10 ng/mL) and dying/dead cells (DAPI; 0.1 μg/mL). Density plot overlay shows nanoplex association within the viable cell population (PE-A/APC-A; right column) (A). Percentage of cells containing LNPs and/or siRNA are displayed as Q1 (LNP positive; LNP⁺siJAK1⁻), Q2 (double positive; LNP⁺siJAK1⁺), Q3 (siJAK1-positive; LNP⁻siJAK1⁺), and Q4 (double negative; LNP⁻siJAK1⁻) cell fractions, summarized in a stacked graph (B). Data is shown as mean ± SD of triplicates of one experiment (n = 3; N = 1).