Journal: STAR Protocols

Article Title: Isolation and culture of mouse alveolar type II cells to study type II to type I cell differentiation

doi: 10.1016/j.xpro.2020.100241

Figure Lengend Snippet:

Article Snippet: Mlg mouse lung fibroblast cells , ATCC , CCL-206.

Techniques: Cell Isolation, Recombinant, SYBR Green Assay, Modification, Red Blood Cell Lysis, Software, Microscopy, Saline, Pore Size, Cell Culture

Journal: Communications biology

Article Title: Macrophage-derived GPNMB trapped by fibrotic extracellular matrix promotes pulmonary fibrosis.

doi: 10.1038/s42003-022-04333-5

Figure Lengend Snippet: Fig. 3 Increased GPNMB protein level was involved in the effect of fibrotic ECM on lung fibroblast activation. a The effect of the GPNMB protein (0.1 μg/mL) on normal fibroblast viability; n ≥5 independent experiments, ***p < 0.001, **p < 0.01. b The effect of GPNMB (0.1 μg/mL) on fibroblast proliferation; n ≥5 independent experiments, *p < 0.05. c, d Immunofluorescence staining was used to evaluate the effect of GPNMB (0.1 μg/mL) on fibroblast proliferation; scale bar = 200 μm, n ≥5 independent experiments, **p < 0.01. e, f The effect of GPNMB (0.1 μg/mL) on fibroblast migration; scale bar = 100 μm, n ≥5 independent experiments, ***p < 0.001. g The effect of GPNMB (0.1 μg/mL) on fibroblast activation; n ≥5 independent experiments, *p < 0.05. h, i The effect of fibrotic ECM and a GPNMB-neutralizing antibody (0.5 μg/mL) on fibroblast migration; scale bar = 100 μm, n ≥5 independent experiments, ***p < 0.001, *p < 0.05. Nor-ECM, normal ECM; Fib-ECM, fibrotic ECM; Ab, GPNMB-neutralizing antibody.

Article Snippet: Mouse lung fibroblast cells (MLg [Mlg 2908] [ATCC® CCL206TM]) were cultured in DMEM containing fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS).

Techniques: Activation Assay, Staining, Migration

Journal: Communications biology

Article Title: Macrophage-derived GPNMB trapped by fibrotic extracellular matrix promotes pulmonary fibrosis.

doi: 10.1038/s42003-022-04333-5

Figure Lengend Snippet: Fig. 5 Macrophage deletion via clodronate liposomes relieved PF and fibrotic ECM affected normal fibroblast behaviors in silicosis mice. a Design of the macrophage deletion experiment. b CT images, H&E staining, and Masson’s trichrome staining of lung tissue; scale bar = 200 μm. c H&E staining and Masson trichrome staining of ECM; scale bar = 200 μm. d, e Changes in cell viability and fibroblast numbers in the fibrotic ECM of silicosis mice after macrophage deletion; n ≥5 independent experiments, ***p < 0.001, **p < 0.01. f, g The effect of macrophage deletion on fibroblast migration induced by fibrotic ECM; scale bar = 200 μm, n ≥5 independent experiments, **p < 0.01. NS, normal saline.

Article Snippet: Mouse lung fibroblast cells (MLg [Mlg 2908] [ATCC® CCL206TM]) were cultured in DMEM containing fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS).

Techniques: Liposomes, Staining, Migration, Saline

Journal: Communications biology

Article Title: Macrophage-derived GPNMB trapped by fibrotic extracellular matrix promotes pulmonary fibrosis.

doi: 10.1038/s42003-022-04333-5

Figure Lengend Snippet: Fig. 7 The CD44/Serpinb2 pathway is related to changes in fibroblast function driven by fibrotic ECM. a Heatmap of differentially expressed mRNAs between fibroblast in different types of ECM. b Volcano plot of differentially expressed proteins between fibroblasts in different types of ECM; |log2(fc)| > 1, p < 0.05. c The KEGG pathways analysis of the upregulated mRNAs. d The GO enrichment analysis of the upregulated mRNAs. e Protein interaction network analysis between GPNMB and Serpinb2. f The change in Serpinb2 protein levels after GPNMB treatment; n ≥5 independent experiments. g The expression level of CD44 gene in different groups on the spatial plots. h The change in CD44 levels in the four groups, as determined by spatial transcriptomics. i CD44 protein levels in fibroblasts after TGFβ1 (5 ng/mL) treatment; n ≥5 independent experiments. NS, normal saline; Nor-ECM, normal ECM; Fib- ECM, fibrotic ECM.

Article Snippet: Mouse lung fibroblast cells (MLg [Mlg 2908] [ATCC® CCL206TM]) were cultured in DMEM containing fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS).

Techniques: Expressing, Saline

Journal: bioRxiv

Article Title: GRAMD2 + alveolar type I cell plasticity facilitates cell state transitions in organoid culture

doi: 10.1101/2023.10.17.560801

Figure Lengend Snippet: A. Organoid formation of FACS-sorted GFP + cells from Gramd2 creERT2;mTmG mice co-cultured with Mlg fibroblasts in Matrigel. Day 6, Day 13, Day 17, Day 19 and Day 25 indicate the number of days in culture post-seeding in Matrigel. N=6. B. Representative whole mount confocal IF images (left) and quantification (right) of organoids formed in Matrigel from GFP + Gramd2 creERT2;mTmG cells. Green = GFP, red = AQP5, purple = proSFTPC. Scale bar = 20 μm. N=3. C. Representative confocal images (left) and quantification (right) of proSFTPC + GFP + paraffin-embedded organoid sections. Green = GFP, red = proSFTPC, blue = DAPI. Scale bar = 20 μm. N=3. D. Representative confocal images of paraffin-embedded organoid sections co-stained for GFP (green), AQP5 (red), proSFTPC (purple), and DAPI (blue) on Day 19. Scale bar = 20 μm (left), 10 μm (right). White arrows indicate GFP + AQP5 + proSFTPC + cells, blue arrows indicate GFP + AQP5 + proSFTPC - cells. E. Representative whole mount confocal images of organoids immunostained for GFP (red), AQP5 (purple) and EdU (green) on Day 13 and Day 25 post-cell seeding. Scale bar = 20 μm. N=3.

Article Snippet: Sorted GFP + cells (12,000–18,000) were seeded with 100,000 Mlg fibroblasts (#CCL 206, American Type Culture Collection, Manassas, VA) in growth factor-reduced Matrigel (#354230, Corning, Glendale, AZ) on Transwell filter inserts (#3470, Corning) in Dulbecco’s DMEM/F-12 (#D6421, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (#SH30070.03, Hyclone, GE Healthcare Life Sciences, Marlborough, MA), 10 mg/l insulin, 5.5 mg/l transferrin, 2 mM glutamine, 100 units penicillin/streptomycin and 0.01 mM SB431542 (#1614/1, R&D systems).

Techniques: Cell Culture, Staining