Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal stromal cell‐secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes

doi: 10.1002/sctm.21-0058

Figure Lengend Snippet: Effect of mesenchymal stromal cell (MSC) condition medium (CM) on antimicrobial properties of primary equine keratinocytes. Keratinocytes were prestimulated with Dulbecco's modified Eagle medium (DMEM), CM from MSCs, or dermal fibroblast (NBL6) CM for 24 hours. CM from these stimulated keratinocytes was collected and added to methicillin‐susceptible Staphylococcus aureus ( MSSA ) or methicillin‐resistant Staphylococcus aureus ( MRSA ) bacterial cultures for 8 hours, after which relative bacterial growth was assessed by optical density (OD). Different letters indicate statistically significant difference. n = 3. (A) Standard curve showing the correlation between CFU/mL and OD readings for both MSSA and MRSA (B)

Article Snippet: The equine dermal fibroblast cell line NBL6 (ATCC, Manassas, Virginia) was cultured in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Modification

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal stromal cell‐secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes

doi: 10.1002/sctm.21-0058

Figure Lengend Snippet: Effect of MSC CM on AMP expression in primary equine keratinocytes. Keratinocytes were incubated with Dulbecco's modified Eagle medium (DMEM), MSC CM, or dermal fibroblasts (NBL6) CM for 24 hours and AMP expression was visualized using IF stainings. Images were collected via confocal microscopy, analyzed, and normalized to cell count using Fiji ImageJ. Different letters indicate statistically significant differences. n = 3 (i). Representative images of AMP expression (green) in DMEM, MSC CM, or NBL6 CM‐stimulated keratinocytes. DAPI was used for nuclear staining (blue) to determine cell number (ii). AMP, antimicrobial peptide; CM, condition medium; DAPI, 4′,6‐diamidino‐2‐phenylindole; MSC, mesenchymal stromal cell

Article Snippet: The equine dermal fibroblast cell line NBL6 (ATCC, Manassas, Virginia) was cultured in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Expressing, Incubation, Modification, Confocal Microscopy, Cell Counting, Staining

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal stromal cell‐secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes

doi: 10.1002/sctm.21-0058

Figure Lengend Snippet: Mesenchymal stromal cells (MSCs) secrete chemokines with pro‐inflammatory effect on keratinocytes. A, Conditioned medium (CM) from MSCs and dermal fibroblasts (NBL6) were submitted for analysis of secreted cytokines and chemokines using equine‐specific immunoassays. Whereas several cytokines were not detected (i), the chemokines CCL2, CCL5, and CCL11 were found to be present in higher concentrations in MSC CM compared to NBL6 CM (ii). Asterisks indicate statistical significance ( P < .05), nd = not detectable, ns = not statistically significant. B, Equine keratinocytes express CCR3 and CCR4 receptors as visualized using immunofluorescence (IF) staining, with CCR expression in green and DAPI used as nuclear staining (blue). C,D, Chemokines CCL2, CCL5, and CCL11 present in MSC CMs were blocked by preincubation with specific equine anticytokine antibodies for 24 hours. Untreated MSC CM and CM treated with an equine IgG1 isotype control were included as controls. MSC CMs were then used to stimulate equine primary keratinocytes and ß‐defensin expression was determined in these keratinocytes (C) or CM was collected from these cells and used in a MRSA growth assay (D). Dotted lines indicate ß‐defensin expression in equine primary keratinocytes stimulated with DMEM (C) and MRSA growth in keratinocyte CM from keratinocytes stimulated with DMEM (D). + and − indicate presence and absence of antibody in MSC CM, respectively. Different letters indicate statistically significant differences. n = 3. DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; MRSA , methicillin‐resistant Staphylococcus aureus

Article Snippet: The equine dermal fibroblast cell line NBL6 (ATCC, Manassas, Virginia) was cultured in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Immunofluorescence, Staining, Expressing, Control, Growth Assay, Modification