Fig. 2: FACS isolation and culture of lung retinoid-containing cells.
A gating strategy for sorting live, single, UV-positive cells (defined by the enclosed area) using emission at λ = 455 nm upon excitation at λ = 350 nm from lung cell suspensions isolated from Lrat+/+ (a) and Lrat−/− (b) mice. c Isolated retinoid-containing (UV-positive) cells cultured 12 h (left microphotograph, 40X magnification) or 72 h (right microphotograph, 40X magnification); Dashed arrows indicate elongated cell morphology and solid arrows highlight intracellular lipid droplets. d Total retinyl ester concentrations (ng/ng DNA) present in UV-positive cells determined by HPLC after 0, 0.5 and 3 days of culture. Values marked with different letters (a–c) are statistically different (a is different from b, p = 0.0222; a is different from c, p = 0.0031). Statistical differences were first analyzed by a one-way ANOVA followed by multiple comparisons employing Tukey’s HSD post hoc test. All values are given as the mean ± 1 S.D., n = 3 for each group. e Lrat mRNA expression determined by qRT-PCR after 0, 0.5 and 3 days of culture. Values marked with different letters (a, b) are statistically different (a is different from b, p = 0.012). Statistical differences were first analyzed by a one-way ANOVA followed by multiple comparisons employing Tukey’s HSD post hoc test. All values are given as the mean ± 1 S.D., n = 3 for each group, n = 10 for Lrat mRNA expression on day 0. f Acta2 mRNA expression determined by qRT-PCR after 0, 0.5 and 3 days of culture. Values marked with different letters (a, b) are statistically different (a is different from b, p = 0.002). Statistical differences were first analyzed by a one-way ANOVA followed by multiple comparisons employing Tukey’s HSD post hoc test. All values are given as the mean ± 1 S.D., n = 3 for each group, n = 10 for Acta2 mRNA expression on day 0.
