Journal: bioRxiv

Article Title: Mesenchymal Stromal Cell Aging Impairs the Self-Organizing Capacity of Lung Alveolar Epithelial Stem Cells

doi: 10.1101/2021.03.05.434121

Figure Lengend Snippet: ( A ) β-galactosidase activity assay. Young and aged L-MSCs were plated in 6-well dishes at a density of 150,000 L-MSCs/well. L-MSCs were allowed to attach overnight and stained for β-galactosidase activity (scale bar = 50µm). ( B ) Lipofuscin granules were detected in the alveolosphere paraffin sections by Sudan Black B staining. Sections were also stained with nuclear fast red for contrast (scale bar = 20µm). ( C ) Cytokine array. 150,000 young and aged L-MSCs were plated in each well of a 6-well culture dish and grown overnight. Cells were washed with PBS and cultured for 24 hrs in serum-free media (SFM; 1.5 ml/well). The culture media were pooled for each cell type and centrifuged to remove any cell and debris. 1 ml of supernatant was applied to antibody array, dotted with antibodies against 111 mouse cytokines and growth factors in duplicates. ( D ) Antibody array showing comparative expression of cytokines and growth factors in the culture media obtained from young and aged L-MSCs. Cytokines and growth factors showing significant difference are numbered and circled. ( E ) Signal intensity was determined for each dot using Image Quant array analysis software; mean signal intensity was calculated for each cytokine, and plotted. Twelve proteins with statistically significant difference (n = 6; p < 0 . 05 ; unpaired T-test) between the young and aged L-MSCs are shown. Data presented here include pooled data from 3 independent experiments. Proteomics/mass-spectrometry analysis. Cell culture media were collected from young and aged L-MSCs (10 6 cells) after 24 hrs of growth in SFM in 10 cm dishes, and subjected to proteomics analysis by liquid chromatography-mass spectrometry (LCMS). ( G ) A three-dimensional principal component analysis (PCA) plot showing replicated samples (young and aged) are relatively similar in their protein expression profiles and grouped together. ( H ) Heat map showing comparative expression of highly secreted proteins in the culture media between young and aged L-MSCs (n = 4). ( I ) The top 36 proteins showing statistically significant difference (n = 4; p < 0 . 05 ; unpaired T-test) between the young and aged L-MSC secretome are plotted.

Article Snippet: Cytokine array was carried out in the young and aged L-MSC culture media using Proteome Profiler Mouse XL Cytokine Array kit (R&D Systems) as per manufacturer ‘s instructions.

Techniques: Activity Assay, Staining, Cell Culture, Ab Array, Expressing, Software, Mass Spectrometry, Liquid Chromatography