normal humanepidermal keratinocytes nheks  (PromoCell)

Journal: Nature Communications

Article Title: Epidermal stem cells control periderm injury repair via matrix-driven specialization of intercellular junctions

doi: 10.1038/s41467-025-64040-7

Figure Lengend Snippet: A Representative immunofluorescence images of P63, F-actin, and DAPI in both layers of the bilayer normal human epidermal keratinocytes (NHEK). DAPI image is z-depth color-coded. B Quantification of P63 intensity (two-tailed unpaired t-test, p < 0.0001, n = 15 (Basal) and 16 (Suprabasal) containing 5–20 cells/FOV (fields of view); 1 independent experiment). Box plot whiskers represent the minimum and maximum value. Bounds mark the 25 th and 75 th percentiles. Central line is the median. C Schematic of gel constructs containing 100% collagen (Collagen) and 100% collagen gel coated with laminin (Col (LM-coated)). D Representative immunofluorescence z-stack cross sections of NHEKs cultured on each gel system. E Representative immunofluorescence images of β-Catenin (CTNNB1) in culture on each gel type. Images of the Suprabasal, Interlayer (Suprabasal-Basal), and Basal shown. Intensity displayed using a 16-color scale. F Quantification of CTNNB1 intensity at junctions for Suprabasal, Interlayer, and Basal in NHEKs cultured on each gel system. For each graph, intensity was normalized to the Collagen Gel Group (two-tailed unpaired t-test (Suprabasal and Basal) and two-tailed Mann-Whitney test (Interlayer), p = 0.0046 (Suprabasal), 0.2213 (Interlayer), and p < 0.0001 (Basal), n = Suprabasal: 44 (Collagen) and 51 (Col LM coated) cells, Interlayer: 53 cells (Collagen and Col LM coated), and Basal: 41 (Collagen) and 44 (Col LM-coated) cells; 1 independent experiment). Data presented as box plots with whiskers to minimum and maximum value. Bounds mark the 25 th and 75 th percentiles. Central line is the median. G Representative immunofluorescence images of Desmoplakin (DSP) in culture on each gel type. Layers and intensity displayed as in ( E ). H Quantification of Desmoplakin intensity at junctions as in ( F ). (two-tailed unpaired t-test (Suprabasal and Interlayer) and two-tailed Mann-Whitney test (Basal), p = 0.0042 (Suprabasal) and p < 0.0001 (Interlayer and Basal), n = Suprabasal: 41 (Collagen) and 42 (Col LM coated) cells, Interlayer: 48 (Collagen) and 50 (Col LM coated) cells, and Basal: 40 (Collagen) and 42 (Col LM-coated) cells; 1 independent experiment). Data presented as box plots with whiskers to minimum and maximum value. Bounds mark the 25 th and 75 th percentiles. Central line is the median. Source data are provided as a Source Data file.

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were purchased from Promo Cell (C-12001) from a 4-year-old Caucasian male donor (Lot #: 494Z030.1).

Techniques: Immunofluorescence, Two Tailed Test, Construct, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Enhanced Differentiation Potential of Pigmented Human Epidermal Equivalents

doi: 10.1101/2025.05.29.656859

Figure Lengend Snippet: (A) Desmosomes (white arrows) connecting adjacent keratinocytes (KER). NM, nuclear membrane. Scale bar: 5 µm. (B) Melanin organelles are found in the perinuclear area of the KER, between the nuclear membrane (NM) and the keratin bundles (white arrowheads). Magnified picture of the boxed area is shown in the right panel. Scale bars: 2 µm (left) and 1 µm (right). (C) Melanocore (MC, thin black arrow) exocytosed from a melanocyte dendrite (MD) docking into KER’s plasma membrane. Magnified picture of the boxed area is shown in the right panel. Scale bars: 2 µm (left) and 0.5 µm (right) .

Article Snippet: Human primary epidermal keratinocytes (#C-12001; PromoCell, Heidelberg, Germany) were maintained in keratinocyte growth medium 2 (#C-20011, PromoCell, Heidelberg, Germany) up to 90% of confluence.

Techniques: Membrane, Clinical Proteomics

Journal: bioRxiv

Article Title: Enhanced Differentiation Potential of Pigmented Human Epidermal Equivalents

doi: 10.1101/2025.05.29.656859

Figure Lengend Snippet: (A) UMAP projection illustrating major cell types and identified keratinocyte subpopulations. (B) UMAP plots split by study, illustrating cell distribution across samples. (C) Violin plots showing the expression of predefined skin marker genes across identified cell populations. BAS, basal keratinocytes; SPN, spinous keratinocytes; GRN, granular keratinocytes; Fib, fibroblasts; MEL, melanocytes; HF, hair follicle-associated cells; LC, Langerhans cells; Endo, endothelial cells.

Article Snippet: Human primary epidermal keratinocytes (#C-12001; PromoCell, Heidelberg, Germany) were maintained in keratinocyte growth medium 2 (#C-20011, PromoCell, Heidelberg, Germany) up to 90% of confluence.

Techniques: Expressing, Marker

Journal: bioRxiv

Article Title: Enhanced Differentiation Potential of Pigmented Human Epidermal Equivalents

doi: 10.1101/2025.05.29.656859

Figure Lengend Snippet: (A) UMAP visualization of subsetted keratinocyte cell states. Contributions of each organoid to the overall UMAP is represented on the right. (B) Heatmap of MetaNeighbor analysis showing AUROC-based similarity across keratinocyte states. The red box highlights PmtHEE-associated suprabasal cell states, while the green box outlines PmtHEE-associated basal cell states. (C) Bar plot showing the proportion of keratinocyte cell states across different skin models. BAS, basal; SPN, spinous; GRN, granular.

Article Snippet: Human primary epidermal keratinocytes (#C-12001; PromoCell, Heidelberg, Germany) were maintained in keratinocyte growth medium 2 (#C-20011, PromoCell, Heidelberg, Germany) up to 90% of confluence.

Techniques:

Journal: bioRxiv

Article Title: Enhanced Differentiation Potential of Pigmented Human Epidermal Equivalents

doi: 10.1101/2025.05.29.656859

Figure Lengend Snippet: (A) Pseudotime-based prediction of cell transition paths. (B) Comprehensive summary of lineage predictions based on identified keratinocyte (KC) states.

Article Snippet: Human primary epidermal keratinocytes (#C-12001; PromoCell, Heidelberg, Germany) were maintained in keratinocyte growth medium 2 (#C-20011, PromoCell, Heidelberg, Germany) up to 90% of confluence.

Techniques:

Journal: bioRxiv

Article Title: Enhanced Differentiation Potential of Pigmented Human Epidermal Equivalents

doi: 10.1101/2025.05.29.656859

Figure Lengend Snippet: (A) Schematic illustration of cell-cell communication across different skin models. (B) Venn diagram showing the predicted ligands under different culture conditions, based on exFINDER analysis. ’FsEpi_MEL’ represents ligands derived from melanocytes in in vivo skin; ’PmtHEE_env’ refers to ligands from the external environment (including melanocytes) in the PmtHEE model; and ’FibHSE_env’ denotes ligands from the external environment in the FibHSE culture model. Keratinocytes were used as the target cells in all analyses. (C) Heatmap of the expression levels of components in the L1CAM-EZR-driven exSigNet under FsEpi and PmtHEE conditions. Shared components are highlighted in red. (D) Topology plot showing the shared L1CAM-EZR-driven exSigNet, constructed using only shared genes. Repeated target genes were marked in red. (E) Violin plot showing the expression of ligand-receptor-transcription factor components from the shared L1CAM-EZR-driven exSigNet.

Article Snippet: Human primary epidermal keratinocytes (#C-12001; PromoCell, Heidelberg, Germany) were maintained in keratinocyte growth medium 2 (#C-20011, PromoCell, Heidelberg, Germany) up to 90% of confluence.

Techniques: Derivative Assay, In Vivo, Expressing, Construct

Journal: bioRxiv

Article Title: Enhanced Differentiation Potential of Pigmented Human Epidermal Equivalents

doi: 10.1101/2025.05.29.656859

Figure Lengend Snippet: (A) Violin plot showing the expression of ligand-receptor-transcription factor components from the shared L1CAM-EZR-driven exSigNet, predicted between FsEpi and PmtHEE. The FibHSE model is now included to enable comprehensive comparison across all conditions. (B) Functional enrichment analysis of biological processes in keratinocytes from FibHSE and PmtHEE. (C) Heatmap showing the expression levels of components within the fibroblast- and environment-specific exSigNets identified in the FibHSE model. Due to substantial overlap of target genes with those in , only ligand-receptor-transcription factor components are presented here. Transcription factors shared with the FsEpi/PmtHEE-derived L1CAM-EZR-driven exSigNet are highlighted in red. ’FibHSE_Fib’ represents ligands derived from fibroblasts in the FibHSE model; ’FibHSE_env’ refers to ligands from the external environment in the FibHSE model. Keratinocytes were used as the target cells in all analyses. (D) Schematic illustrating the origin of alternative exSigNets in the FibHSE model that bypass the L1CAM-EZR-driven exSigNet, in contrast to the melanocyte-to-keratinocyte signaling observed in FsEpi and PmtHEE.

Article Snippet: Human primary epidermal keratinocytes (#C-12001; PromoCell, Heidelberg, Germany) were maintained in keratinocyte growth medium 2 (#C-20011, PromoCell, Heidelberg, Germany) up to 90% of confluence.

Techniques: Expressing, Comparison, Functional Assay, Derivative Assay

Journal: Frontiers in Aging

Article Title: The aldose reductase inhibitors AT-001, AT-003 and AT-007 attenuate human keratinocyte senescence

doi: 10.3389/fragi.2024.1466281

Figure Lengend Snippet: Senescence induction upregulates AR activity. AR activity measured by colorimetric assay in human keratinocytes either treated with (A) 10 uM H 2 O 2 or Vehicle (Veh- dH 2 O) (B) 25 mM high glucose (HG) or normal glucose (5 mM) (NG) for 48 h (C) 200 nM Mitomycin-C (MMC) for 24 h compared to Veh (DMSO) and (D) 0.2 nM AT-001 for 48 h vs. Veh (1:5,000 dilution DMSO in PBS). Data are presented as the mean ± SEM. n = 4 per group obtained from two consecutive experiments. Unpaired t-test was implemented for comparison between two groups. p* < 0.05 vs. Veh or NG.

Article Snippet: Normal pooled adult Human Epidermal Keratinocytes (NHK) were obtained from PromoCell (cat # C-12006) maintained and passaged in serum free Keratinocyte complete growth medium (PromoCell, cat # C-20111).

Techniques: Activity Assay, Colorimetric Assay, Comparison

Journal: Frontiers in Aging

Article Title: The aldose reductase inhibitors AT-001, AT-003 and AT-007 attenuate human keratinocyte senescence

doi: 10.3389/fragi.2024.1466281

Figure Lengend Snippet: ARIs prevent HG induced senescence phenotype. Human keratinocytes were treated with 25 mM HG or normal condition medium NG. (A) SA-β-gal staining was performed to assess positive senescent cells. (B) qPCR analysis for confirming gene expression of senescent markers CDKN1A, TP53 and SERPINE1 . (C) Skin Cells stained with 5 µM DiHydroethidium (DHE) dye for 30 min to detect cytosolic superoxide and (D) cells stained with 5 µM MitoSOX dye for 10 min to detect mito superoxide. Data are presented as the mean ± SEM. n = 4 per group obtained from two consecutive experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was implemented for comparison between groups. p* < 0.05 vs. NG. p# < 0.05 vs. Veh.

Article Snippet: Normal pooled adult Human Epidermal Keratinocytes (NHK) were obtained from PromoCell (cat # C-12006) maintained and passaged in serum free Keratinocyte complete growth medium (PromoCell, cat # C-20111).

Techniques: Staining, Expressing, Comparison

Journal: Frontiers in Aging

Article Title: The aldose reductase inhibitors AT-001, AT-003 and AT-007 attenuate human keratinocyte senescence

doi: 10.3389/fragi.2024.1466281

Figure Lengend Snippet: ARIs prevent H2O2 induced senescence phenotype. Human keratinocytes were treated with 10 µM H 2 O 2 or Veh (dH 2 O) and ARIs for 48 h. (A) SA-β-gal staining was performed to assess positive senescent cells. (B) Skin Cells stained with 5 µM DiHydroethidium (DHE) dye for 30 min to detect cytosolic superoxide and (C) cells stained with 5 µM MitoSOX dye for 10 min to detect mito superoxide. (D) qPCR analysis for confirming gene expression of senescent markers CDKN1A, TP53 and SERPINE1 . (E) Gamma H2A.X Staining and (F) CellEvent™ Senescence Green Detection staining in human keratinocytes treated with Veh (dH 2 O), 400 µM H 2 O 2 for 1 h followed by assessment of cells at day 10 with or without ARI AT-001. (G) ELISA to measure cytokine release in condition medium collected at day 10 and (H) qPCR analysis for confirming gene expression of senescent markers CDKN1A, TP53 and SERPINE1 CDKN2A and LMNB1 . Data are presented as the mean ± SEM. n = 4 per group obtained from two consecutive experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was implemented for comparison between groups. p* < 0.05 vs. H 2 O 2 . p# < 0.05 vs. Veh.

Article Snippet: Normal pooled adult Human Epidermal Keratinocytes (NHK) were obtained from PromoCell (cat # C-12006) maintained and passaged in serum free Keratinocyte complete growth medium (PromoCell, cat # C-20111).

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Aging

Article Title: The aldose reductase inhibitors AT-001, AT-003 and AT-007 attenuate human keratinocyte senescence

doi: 10.3389/fragi.2024.1466281

Figure Lengend Snippet: ARIs prevent MMC induced senescence phenotype. Human keratinocytes were treated with 200 nM MMC or Veh (DMSO) and ARIs for 24 h. (A) SA-β-gal staining was performed to assess positive senescent cells. (B) qPCR analysis for confirming gene expression of senescent markers CDKN1A, TP53 and SERPINE1 . (C) Skin Cells stained with 5 µM DiHydroethidium (DHE) dye for 30 min to detect cytosolic superoxide and (D) cells stained with 5 µM MitoSOX dye for 10 min to detect mito superoxide. Data are presented as the mean ± SEM. n = 4 per group obtained from two consecutive experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was implemented for comparison between groups. p* < 0.05 vs. MMC. p# < 0.05 vs. Veh.

Article Snippet: Normal pooled adult Human Epidermal Keratinocytes (NHK) were obtained from PromoCell (cat # C-12006) maintained and passaged in serum free Keratinocyte complete growth medium (PromoCell, cat # C-20111).

Techniques: Staining, Expressing, Comparison

Journal: bioRxiv

Article Title: Human skin rejuvenation via mRNA

doi: 10.1101/2024.11.12.623261

Figure Lengend Snippet: (a) Illustration of Human Skin Aging. Human skin aging is characterized by distinct morphological and functional changes in both the epidermis and dermis. The epidermis becomes noticeably thinner in aged skin due to a reduction in keratinocyte proliferation and an increase in apoptosis. In the dermis, a significant hallmark of aging is the decreased production of collagen by fibroblasts. (b) UMAP visualizations reveal the categorization of human keratinocytes into 11 subtypes. Basal cells (BC), spinous cells (SC), and granule cells (GC) are indicated based on the expression matrix and their specific markers. Each cell type is distinguished by a unique color. (c) Expression profiles of specified marker genes are depicted across different subtypes. Gradations from gray to blue denote varying levels of gene expression, whereas deeper blue signifies higher expression. Granular cells (GC) are characterized by FLG expression, spinous cells (SC) by KRT10, and basal cell subtype 2 (BC2) by KRT5 and KRT14. (d) Heatmap presenting the scaled expression levels of genes prominently expressed exclusively within all BC, SC, and GC subtypes. The color spectrum, progressing from white to blue, signifies low to elevated gene expression levels. (e) Visualization of the lineage development of keratinocytes along differentiation trajectories. (f) Pseudo-time distribution of cells within lineage S0-S1-S3 for both young and old samples, separated by combined aging and chronological aging scenarios.

Article Snippet: Primary human epidermal keratinocytes from a 27-year-old donor (PromoCell, C-12003) and 56-year donor (Lifeline Cell Technology, C-0025) were cultured in Keratinocyte Growth Medium (Lifeline Cell Technology, LL-0007), which was devoid of Gentamicin and Amphotericin B but supplemented with 100U/ml Penicillin-Streptomycin.

Techniques: Functional Assay, Expressing, Marker

Journal: bioRxiv

Article Title: Human skin rejuvenation via mRNA

doi: 10.1101/2024.11.12.623261

Figure Lengend Snippet: (a) Overview summarizing the development of mRNA therapy for skin rejuvenation. (b) Identification of two distinct “gene modules” emerging during the aging process based on single-cell gene expression clustering: one characterized by overexpression and the other by under-expression. Genes within these modules are spatially mapped in the skin atlas and integrated into a gene-gene network. (c) Visualization of gene expression patterns within the identified modules using Uniform Manifold Approximation and Projection (UMAP). The gene-gene interaction networks display connectivity, with node size reflecting the number of connections. Bar plots depict expression changes of hub genes across aging stages. (d) Workflow illustrating siRNA-mediated knockdown of ATF3. (e) Immunofluorescence staining of Ki67 in human epidermal keratinocytes following siRNA-mediated ATF3 knockdown. Scale bar, 125 µm. Data are presented as mean ± SEM. n = 6 per group. * p < 0.05. (f) Quantitative PCR (qPCR) analysis of Senescence-associated Secretory Phenotype (SASP) in human epidermal keratinocytes upon siRNA-mediated ATF3 knockdown. Data are presented as mean ± SEM. (g) Senescence-associated β-galactosidase (SA-β-gal) staining of human epidermal keratinocytes following siRNA-mediated ATF3 knockdown. Scale bar, 125 µm. Data are presented as mean ± SEM. n = 3 per group. ** p < 0.01. (h) Workflow depicting mRNA treatment by overexpressing the ATF3 gene, designed with in-house untranslated regions. (i) Immunofluorescence staining of Ki67 in human epidermal keratinocytes following mRNA treatment of ATF3. Scale bar, 125 µm. Data are presented as mean ± SEM. n = 6 per group. * p < 0.05. (j) qPCR analysis of SASP in human epidermal keratinocytes upon mRNA treatment of ATF3. Data are presented as mean ± SEM. (k) SA-β-gal staining of human epidermal keratinocytes following mRNA treatment of ATF3. Scale bar, 125 µm. Data are presented as mean ± SEM. n = 3 per group. *** p < 0.001.

Article Snippet: Primary human epidermal keratinocytes from a 27-year-old donor (PromoCell, C-12003) and 56-year donor (Lifeline Cell Technology, C-0025) were cultured in Keratinocyte Growth Medium (Lifeline Cell Technology, LL-0007), which was devoid of Gentamicin and Amphotericin B but supplemented with 100U/ml Penicillin-Streptomycin.

Techniques: Expressing, Over Expression, Knockdown, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Human skin rejuvenation via mRNA

doi: 10.1101/2024.11.12.623261

Figure Lengend Snippet: (a) Expression levels of ATF3 in young and old skin samples from our dataset. (b) Expression levels of ATF3 in young and old skin samples from other datasets. (c) Immunofluorescence staining of ATF3 in human skin. Scale bar, 200 mm; n = 3 for each group. (d) Quantitative PCR (qPCR) analysis of Senescence-associated Secretory Phenotype (SASP) and senescence-associated β-galactosidase (SA-β-gal) staining of human epidermal keratinocytes upon siRNA-mediated gene knockdown. Data are presented as mean ± SEM. n = 3 per group. **p < 0.01. (e) Comparison of senescence levels in human epidermal keratinocytes treated with mCherry Control and ATF3. The experiments were conducted in triplicate. Senescence-associated β-galactosidase (SA-β-gal) staining using C12FDG revealed significantly lower senescence levels in ATF3-treated cells compared to mCherry-treated controls. Data are presented as mean ± SD, with statistical significance determined by t-test. (f) Dose-response curve of ATF3 mRNA on senescence levels in human epidermal keratinocytes. Five concentrations were tested, and SA-β-gal activity was measured. Data are presented as mean ± 95% confidence interval. (g) mRNA treatment with the P2A structure of ATF3 mRNA. Senescence levels and mCherry positivity were assessed in human epidermal keratinocytes treated with mCherry controls and ATF3 P2A samples. Senescence levels were evaluated using SA-β-gal staining with C12FDG. Data are presented as mean ± SD and statistical significance was determined by t-test. ****p < 0.0001. (h) qPCR analysis of ATF3 mRNA expression levels in keratinocyte with and without exogenous ATF3 mRNA delivery. Data are presented as mean ± SEM. n = 3 per group. ****p < 0.0001.

Article Snippet: Primary human epidermal keratinocytes from a 27-year-old donor (PromoCell, C-12003) and 56-year donor (Lifeline Cell Technology, C-0025) were cultured in Keratinocyte Growth Medium (Lifeline Cell Technology, LL-0007), which was devoid of Gentamicin and Amphotericin B but supplemented with 100U/ml Penicillin-Streptomycin.

Techniques: Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Knockdown, Comparison, Control, Activity Assay

Journal: bioRxiv

Article Title: Human skin rejuvenation via mRNA

doi: 10.1101/2024.11.12.623261

Figure Lengend Snippet: (a) Mechanistic insights into skin aging mediated by secreted proteins from keratinocytes to fibroblasts. (b) Network representation illustrating the crosstalk between different cell types, with edge width proportional to the number of identified ligand-receptor pairs. Numbers on edges indicate significant ligand-receptor interactions between cell populations. (c) Significant ligand-receptor pairs contributing to signaling from keratinocytes to fibroblasts were assessed using one-sided permutation tests ( p -values). (d) Expression profiles of secreted proteins from keratinocytes involved in signaling to fibroblasts. (e, f, g, h) Experimental setup involving culture of fibroblasts with conditioned medium from young and old keratinocytes. Evaluation includes Ki67 staining, senescence staining, qPCR of collagen expression, and ELISA of collagen in fibroblasts. Scale bar, 125 µm. Data are presented as mean ± SEM. n = 3 per group. * p < 0.05, ** p < 0.01, ****p < 0.0001. (i, j, k, l) Experimental setup involving culture of fibroblasts with conditioned medium from control and ATF3 treated keratinocytes. Evaluation includes Ki67 staining, senescence staining, qPCR of collagen, and ELISA of collagen in fibroblasts. Scale bar, 125 µm. Data are presented as mean ± SEM. n = 3 per group. * p < 0.05, ** p < 0.01, ***p < 0.001.

Article Snippet: Primary human epidermal keratinocytes from a 27-year-old donor (PromoCell, C-12003) and 56-year donor (Lifeline Cell Technology, C-0025) were cultured in Keratinocyte Growth Medium (Lifeline Cell Technology, LL-0007), which was devoid of Gentamicin and Amphotericin B but supplemented with 100U/ml Penicillin-Streptomycin.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Human skin rejuvenation via mRNA

doi: 10.1101/2024.11.12.623261

Figure Lengend Snippet: (a) The circo plot illustrates potential cell interactions predicted by CellphoneDB among nine major cell types in both combined aging and chronological aging. These cell types include keratinocytes, fibroblasts, endothelial cells, hair follicles, sebocytes, melanocytes, pericytes, immune cells, and Langerhans cells. The size of each node represents the number of interactions, while the width of each edge indicates the number of significant ligand-receptor pairs between the two cell types. (b) The gene-gene network displays potential ligand-receptor pairs between keratinocytes and fibroblasts. (c) The network of gene-gene interactions among down-regulated genes and secreted genes in keratinocytes, as well as the predicted gene-gene connections between keratinocytes and fibroblasts from CellphoneDB. (d) The gene expression profiles of secreted proteins in keratinocytes, involved in the predicted significant ligand-receptor pairs between keratinocytes and fibroblasts, are shown in different samples. (e) Workflow of the Crosstalk Experiment. Keratinocytes were cultured in a basal medium for 24 hours to produce a conditioned medium. This conditioned medium was then mixed 1:1 with fibroblast growth medium and used to culture human fibroblast cells for 72 hours, with fresh conditioned medium replaced every 24 hours. Young KCM, old KCM, control mRNA-treated KCM, and ATF3 mRNA-treated KCM were prepared and utilized for fibroblast culture. After 72 hours, measurements were taken for β-galactosidase activity, collagen and elastin mRNA levels by qPCR, and collagen levels by ELISA.

Article Snippet: Primary human epidermal keratinocytes from a 27-year-old donor (PromoCell, C-12003) and 56-year donor (Lifeline Cell Technology, C-0025) were cultured in Keratinocyte Growth Medium (Lifeline Cell Technology, LL-0007), which was devoid of Gentamicin and Amphotericin B but supplemented with 100U/ml Penicillin-Streptomycin.

Techniques: Expressing, Cell Culture, Control, Activity Assay, Enzyme-linked Immunosorbent Assay