penicillin streptomycin  (PromoCell)

Journal: Disease Models & Mechanisms

Article Title: An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium

doi: 10.1242/dmm.052601

Figure Lengend Snippet: Characterization of immortalized cardiac fibroblasts from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model. (A) Representative flow cytometry plots showing the identification of RFP + cardiac fibroblast subsets. (B) Representative flow cytometry plots for identification of GFP + subsets. (C-F) qRT-PCR analysis of the indicated genes in immortalized cardiac cells from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice. Compared with heart ventricles or immortalized epicardial cells (Epi), immortalized fibroblasts exhibited abundant expression of CF marker genes (C) and almost undetectable expression of marker genes specific to cardiomyocytes (D), mural cells (smooth muscle cells and pericytes) (E) and epicardial cells (F). Data are presented as mean±s.e.m. ( n =3-6); ** P <0.01, *** P <0.001, **** P <0.0001, two-tailed Student’s t -test. (G) Schematic representation of the FACS-based isolation of four fibroblast subpopulations defined by CD90 and GFP expression. Created in BioRender by Martıńez-Estrada, O. M. (2025). https://BioRender.com/h3kmkr0 . (H) qRT-PCR analysis of Acta2 expression in these subsets revealed elevated Acta2 levels in the CD90 + GFP − population. Data are presented as mean±s.e.m. ( n =6); **** P <0.0001, one-way ANOVA followed by Tukey's post-hoc test.

Article Snippet: To assess the impact of fibroblast medium on cellular phenotype and stability of marker gene expression, cells were cultured in a commercially available fibroblast medium (Promocell, C-23025) for three passages, followed by subsequent analyses.

Techniques: Flow Cytometry, Quantitative RT-PCR, Expressing, Marker, Two Tailed Test, Isolation

Journal: Disease Models & Mechanisms

Article Title: An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium

doi: 10.1242/dmm.052601

Figure Lengend Snippet: TGFβ induces a profibrotic phenotype in immortalized RFP + cardiac fibroblasts. (A) qRT-PCR analysis of Acta2 expression in immortalized RFP + cardiac fibroblasts maintained in DMEM or fibroblast medium (FM). Data are presented as mean±s.e.m. ( n =4-5); **** P <0.0001, two-tailed Student’s t -test. (B) Representative flow cytometry plots of immortalized RFP + cardiac fibroblasts cultured in FM. (C) Quantification of CD90/GFP fibroblast subsets (CD90 + GFP + , CD90 + GFP − , CD90 − GFP + , CD90 − GFP − ) in DMEM versus FM. Data are presented as mean±s.e.m. ( n =3), ** P <0.01, **** P <0.0001, two-tailed Student’s t -test. (D,E) Western blot and densitometric analysis of phosphorylated SMAD2 (pSMAD2) in immortalized cardiac fibroblasts treated with TGFβ, with total SMAD2 serving as the loading control. Data are presented as mean±s.e.m. ( n =3); **** P <0.0001, one-way ANOVA followed by Tukey's post-hoc test. (F) qRT-PCR analysis of the indicated genes in immortalized cardiac fibroblasts cultured with TGFβ for 3 days. Data are presented as mean±s.e.m. ( n =3), * P <0.05, ** P <0.01, two-tailed Student’s t -test.

Article Snippet: To assess the impact of fibroblast medium on cellular phenotype and stability of marker gene expression, cells were cultured in a commercially available fibroblast medium (Promocell, C-23025) for three passages, followed by subsequent analyses.

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Flow Cytometry, Cell Culture, Western Blot, Control