Fig 1: Expression of the functionality marker lysozyme and secretory levels of the active lysozyme enzyme NAG on day 40 of differentiation (A) IF staining of Lysozyme+/Na+/K + ATPase lacrimal gland acinar cells (Figures are representative of three independent differentiation experiments, scale bar: 40 µm) (B) NAG concentration in the supernatant of lacrimal gland organoids following carbachol and forskolin stimulation (Two-Way ANOVA. Error bars represent standard error of mean. ***p ≤ .001; ****p ≤ .0001, n = 3 biological replicates). (C) Swelling of organoids following carbachol and forskolin stimulation (Unpaired t-test. Error bars represent standard error of mean. *p ≤ .05, n = 4 biological replicates).
Fig 2: Analyses of implanted cells.a Immunofluorescence imaging using LIVE/DEAD™ staining demonstrated high cell viability on cell inserts after 3- and 7-day implants. Quantification of LIVE/DEAD™ staining demonstrated 95.6 ± 1.7 and 90.3 ± 4.5% viability (mean ± standard deviation) of cell inserts after 3- and 7-day implants, respectively (for 3-day implants n = 6 biologically independent samples from 3 animals, for 7-day implants n = 3 independent samples from 2 animals). b Cell inserts after 3- and 7-day implants demonstrated confluency and integrity of the cell monolayer, as examined by immunofluorescence imaging of tight junction protein ZO-1 (green) and DAPI (blue) nuclear staining (for 3-day implants n = 9, for 7-day implants n = 6). c Gene expression of cell transporters AQP1 and NHE3 and vitamin D 1a Hydroxylase were measured using qPCR as a surrogate of renal cell function. Relative to in vitro controls, AQP1 expression was 1.05x ± 0.35 (mean ± standard deviation), while 1a Hydroxylase and NHE3 were 1.56 ± 0.18 and 2.43 ± 0.27 x more expressed (n = 4 biologically independent samples). d The activity of damage biomarkers NAG and GGT was measured after days 3 and 7, using ELISA and normalized to in vitro controls. HREC from implants demonstrated NAG expression levels similar to those of in vitro controls and were significantly lower than a standard positive control (0.93 ± 0.15 for day 3) (2.01 ± 0.77 for day 7) (mean values ± standard deviation). Day 7 GGT levels were comparable to in vitro control cells and significantly lower than a standard positive control (1.37 ± 0.04) (n = 3 biologically independent samples). * or # p < 0.05, ** or ## p < 0.001, where * is for day 3 comparisons and # is for day 7. Source data are provided as a Source Data file.
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