Fig. 2.

Detection, quantification, and isolation of liver myofibroblasts. (A) Strategy to analyze myofibroblasts by flow cytometry: Collagen type I-expressing myofibroblasts were identified in nonparenchymal fraction by GFP expression and further fractionated to Vit.A⁺ and Vit.A⁻ cells. (B) FACS analysis of nonparenchymal fraction from untreated and BDL-, and CCl₄- treated Col-GFP mice: GFP⁺ cells were detected by argon laser at 488 nm wavelength, and Vit.A⁺ cells were detected by violet laser at 405 nm wavelength. Representative dot plots are shown, P < 0.03. GFP⁺Vit.A⁺ and GFP⁺Vit.A⁻ cells were sort purified and analyzed by light and fluorescent microscopy for GFP and Vitamin A expression (UV laser, 20× objective). (C) Flow cytometry-based quantification of GFP⁺ myofibroblasts. Expression of vitamin A in GFP⁺ cells was analyzed in nonparenchymal fraction of Col-GFP mice at different time points (n = 6 per time point) of CCl₄ and BDL, P < 0.01. (D) Immunophenotyping of GFP⁺ myofibroblasts isolated from BDL mice. GFP⁺Vit.A⁺ and GFP⁺Vit.A⁻ fractions were sort purified from Col-GFP mice (n = 6) after BDL (20 d). Expression of myofibroblast marker (α-SMA), HSC markers (desmin, GFAP, CD146), and PF markers (elastin, Thy1) were analyzed by immunocytochemistry using specific antibodies or isotype matched controls (40× objective). GFP⁺Vit.A⁺ and GFP⁺Vit.A⁻ cells were identified as aHSCs and aPFs, respectively. For each fraction, the percent of positively stained cells is calculated (compared with total cells, 100%, P < 0.05). (E) Quantification of GFP⁺Vit.A⁺ and GFP⁺Vit.A⁻ fractions is based on expression of HSC- and PF-specific markers in GFP⁺ myofibroblasts (100%) as detected by immunocytochemistry, P < 0.05.