cardiac fibroblast growth medium  (Cell Applications Inc)

Journal: Biomedicines

Article Title: Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

doi: 10.3390/biomedicines13112829

Figure Lengend Snippet: Impact of cell culture conditions on human cardiac fibroblasts (CFs) morphology and phenotype. ( A ) Morphology of CFs after 48h in conventional 2D culture ( a , c ) and 3D culture on ARdH hydrogel ( b , d ), as determined by F-actin labeling with phalloidin (red) and DAPI nuclear staining (cyan). The scale bar indicates 200 µm for ( a , b ), and 50 µm for ( c , d ). ( B ) Cell viability of CFs after 48 h in 2D and 3D culture, as determined by MTT assay; ( C ) MCP-1 released in conditioned media by CFs cultured in 2D and 3D conditions determined by ELISA assay; ( D ) mRNA expression of α-SMA, MCP-1, and IL-1β in CFs isolated from 3D-hydrogel or 2D culture after 48 h. ( E ) α-SMA protein expression in CFs from aortic root-derived hydrogel (ARdH) or 2D culture, determined by Western Blot; n = 3, * p < 0.05. Data is represented as mean ± SEM.

Article Snippet: Adult human cardiac fibroblasts (CFs) (C-12375) purchased from Merck (Darmstadt, Germany) were sub-cultured using Cardiac Fibroblast Growth Medium (316-500—Cell Applications, San Diego, CA, USA).

Techniques: Cell Culture, Labeling, Staining, MTT Assay, Enzyme-linked Immunosorbent Assay, Expressing, Isolation, Derivative Assay, Western Blot

Journal: Biomedicines

Article Title: Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

doi: 10.3390/biomedicines13112829

Figure Lengend Snippet: Profile of inflammatory mediators and of matrix metalloproteinases (MMP) following a 24 h indirect co-culture of human CFs grown on ARdH hydrogel with control granulocytes (CfN), or their pro-inflammatory (N1) or anti-inflammatory (CfN2) subtypes. ( A ) mRNA level of inflammatory cytokines and chemokines in CFs grown on ARdH hydrogel and indirectly interacted with granulocytes subsets, determined by qPCR—normalized to B2M and represented as fold change compared with C (Cf only); ( B ) Levels of secreted inflammatory cytokines and chemokines in conditioned media from indirect co-cultures as evaluated by ELISA assay; ( C ) IL-1β protein level in CFs lysates following indirect interaction with neutrophil-like cell subtypes, as determined by Western Blot assay and normalized to β-actin; ( D ) Metalloproteases MMP-1, -2, -3, -9 and -13 mRNA expression in CFs indirectly co-cultured with neutrophil-like subtypes; ( E ) Concentration of soluble MMP-1 and MMP-9 quantified in conditioned media as evaluated by ELISA assay; ( F , G ) Protein expression of MMP-9/-1 in CFs following indirect interaction, as evaluated by Western Blot and normalized to β-actin; ( H ) The enzymatic activity of gelatinase MMP-9 in the conditioned media assessed by SDS-PAGE gelatine zymography; ( I ) Intracellular signaling: MAPKs (p38 and ERK1/2) and NF-κB phosphorylation in human CFs following indirect interaction with neutrophil-like cell subtypes; ( J ) Proliferative capacity of primary mouse CFs assessed using the xCelligence system: C+—fibroblasts exposed to DMEM/F12 with 10%FBS and C-—fibroblasts exposed to DMEM/F12 without FBS; N1/N2—fibroblasts exposed to mouse N1/N2 conditioned media; ( K ) Cell index after ~3 days of exposure to N1/N2 conditioned media. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 Data is represented as mean ± SEM.

Article Snippet: Adult human cardiac fibroblasts (CFs) (C-12375) purchased from Merck (Darmstadt, Germany) were sub-cultured using Cardiac Fibroblast Growth Medium (316-500—Cell Applications, San Diego, CA, USA).

Techniques: Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Cell Culture, Concentration Assay, Activity Assay, SDS Page, Zymography, Phospho-proteomics