Journal: Frontiers in Physiology

Article Title: Balancing act: optimizing blue light for melanogenesis while minimizing cellular damage in primary human skin cells

doi: 10.3389/fphys.2024.1513054

Figure Lengend Snippet: Sequential Irradiation and Medium Exchange in Melanocyte Culture ± Keratinocyte-Conditioned Medium for Pro-Melanogenic Enzyme Analysis. Primary keratinocytes (Population 1) were cultured separately on a feeder layer of gamma-irradiated human fibroblasts and irradiated on days 3, 5, and 7 using an LED module with specified light parameters (10 mW/cm 2 , 15 J/cm 2 ). The culture medium was collected prior to irradiation to allow α-MSH accumulation over several hours and because cells were irradiated in PBS to prevent photon absorption and ROS generation. The collected medium was stored at −80°C and later concentrated 10-fold. Melanocytes (Populations 1, 3, and 4), seeded in keratinocyte medium with FGF2, received thawed keratinocyte-conditioned medium at 1X concentration post each irradiation. Three types of conditioned medium were used: CM (keratinocyte medium only), CMK (medium from non-irradiated keratinocytes), and CMiK (medium from irradiated keratinocytes). On day 12, melanocytes were harvested and analyzed via Western blot for TYR and DCT, marking the analysis of pro-melanogenic activity across different light and media conditions. I = Irradiated, NI = non-irradiated, Sham = non-irradiated cell culture, KCM = keratinocyte-conditioned medium, WB = Western blot. This figure was sketched using BioRender.

Article Snippet: Melanocytes were isolated from keratinocytes by culturing the epidermal cells for 2 days in melanocyte medium (Melanocyte Basal Medium [Lonza, Basel, Switzerland]) supplemented with 0.5% fetal bovine serum (HyClone, Wilmington, DE, United States), calcium, bovine pituitary extract, recombinant human basic fibroblast growth factor, recombinant human insulin, hydrocortisone, phorbol myristate acetate, penicillin, and gentamicin, along with 0.1 mg/mL geneticin (G418; Sigma Chemicals, St. Louis, MO, United States).

Techniques: Irradiation, Cell Culture, Concentration Assay, Western Blot, Activity Assay

Journal: Frontiers in Physiology

Article Title: Balancing act: optimizing blue light for melanogenesis while minimizing cellular damage in primary human skin cells

doi: 10.3389/fphys.2024.1513054

Figure Lengend Snippet: Keratinocyte cultures were exposed to various light parameters across three time points: day 3 (white bars), day 5 (gray bars), and day 7 (black bars). (A) Effects of Different Irradiation Parameters on Primary Keratinocyte Viability. This bar graph presents the results of the Alamar Blue (AB) assay conducted on primary keratinocyte monolayers. The x -axis displays different combinations of irradiance (mW/cm 2 ) and fluence (J/cm 2 ) for blue light, with the last combination representing UVA light at 15 mW/cm 2 and 20 J/cm 2 . The y -axis indicates the fold change in cell viability relative to sham control (untreated cells). (B) Alpha-MSH Levels in Keratinocyte Cultures Exposed to Various Light Parameters. This graph shows the fold change in alpha-Melanocyte Stimulating Hormone (α-MSH) levels, measured by ELISA. The x -axis lists the combinations of irradiance (mW/cm 2 ) and fluence (J/cm 2 ) for blue light, with the last combination representing UVA light at 15 mW/cm 2 and 20 J/cm 2 . The y -axis displays the fold change in α-MSH levels compared to the sham control (untreated cells). Error bars represent the standard deviation (SD) among the three distinct cell populations (N = 3) analyzed. Statistically significant differences between days for each treatment condition are marked by crosses ( † for p < 0.05, †† for p < 0.01). Statistical differences between a condition (i.e., day and light treatment) and its respective sham are marked by asterisks (* for p < 0.05, ** for p < 0.01, *** for p < 0.001).

Article Snippet: Melanocytes were isolated from keratinocytes by culturing the epidermal cells for 2 days in melanocyte medium (Melanocyte Basal Medium [Lonza, Basel, Switzerland]) supplemented with 0.5% fetal bovine serum (HyClone, Wilmington, DE, United States), calcium, bovine pituitary extract, recombinant human basic fibroblast growth factor, recombinant human insulin, hydrocortisone, phorbol myristate acetate, penicillin, and gentamicin, along with 0.1 mg/mL geneticin (G418; Sigma Chemicals, St. Louis, MO, United States).

Techniques: Irradiation, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Balancing act: optimizing blue light for melanogenesis while minimizing cellular damage in primary human skin cells

doi: 10.3389/fphys.2024.1513054

Figure Lengend Snippet: Relative protein expression of Tyrosinase (Tyr) and Dopachrome Tautomerase (DCT) normalized to β-actin in non-irradiated and irradiated primary melanocytes cultured with or without keratinocyte-conditioned medium. (A) Tyrosinase Protein Expression: Bar graphs show fold change in Tyr levels in non-irradiated (left) and irradiated (right) melanocytes. Conditions include melanocytes without keratinocyte-conditioned medium (CM), with conditioned medium from non-irradiated keratinocytes (CM-K), and irradiated keratinocytes (CM-iK). Significant increases in Tyr expression were observed in non-irradiated melanocytes exposed to irradiated keratinocyte-conditioned medium (CM-iK) ( p < 0.05). Immunoblot images of Tyr and β-actin are shown below the bar graphs. (B) Dopachrome Tautomerase (DCT) Protein Expression: Bar graphs present the fold change in DCT levels in non-irradiated (left) and irradiated (right) melanocytes. A significant increase in DCT expression is seen in non-irradiated melanocytes exposed to irradiated keratinocyte-conditioned medium ( p < 0.05). Immunoblot images of DCT and β-actin are shown below the bar graphs. Error bars indicate the standard deviation (SD) between the three populations of melanocytes (N = 3) assessed, and p < 0.05 is considered statistically significant.

Article Snippet: Melanocytes were isolated from keratinocytes by culturing the epidermal cells for 2 days in melanocyte medium (Melanocyte Basal Medium [Lonza, Basel, Switzerland]) supplemented with 0.5% fetal bovine serum (HyClone, Wilmington, DE, United States), calcium, bovine pituitary extract, recombinant human basic fibroblast growth factor, recombinant human insulin, hydrocortisone, phorbol myristate acetate, penicillin, and gentamicin, along with 0.1 mg/mL geneticin (G418; Sigma Chemicals, St. Louis, MO, United States).

Techniques: Expressing, Irradiation, Cell Culture, Western Blot, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Balancing act: optimizing blue light for melanogenesis while minimizing cellular damage in primary human skin cells

doi: 10.3389/fphys.2024.1513054

Figure Lengend Snippet: Microscopic images of melanocytes (population 1) cultured in conditioned media from keratinocytes (CM, CMK, CMiK) before lysis for Western blot analysis on day 12. Representative images of melanocytes cultured in CM (keratinocyte medium only), CMK (conditioned medium from non-irradiated keratinocytes), and CMiK (conditioned medium from irradiated keratinocytes) are shown for non-irradiated (left panels) and irradiated (right panels) melanocytes. Images were captured at ×10 magnification. Melanocytes in CM show a healthy morphology with well-defined spindle shapes and minimal pigmentation. Those cultured in CMK exhibit slightly more spindle elongation with no noticeable pigmentation changes. Melanocytes cultured in CMiK display enhanced pigmentation, particularly in irradiated conditions, consistent with increased pro-melanogenic factor activity. These photomicrographs were obtained from live-cell cultures without fixation, eliminating the possibility of formalin or other pigment artifacts. Scale bars = 400 µm.

Article Snippet: Melanocytes were isolated from keratinocytes by culturing the epidermal cells for 2 days in melanocyte medium (Melanocyte Basal Medium [Lonza, Basel, Switzerland]) supplemented with 0.5% fetal bovine serum (HyClone, Wilmington, DE, United States), calcium, bovine pituitary extract, recombinant human basic fibroblast growth factor, recombinant human insulin, hydrocortisone, phorbol myristate acetate, penicillin, and gentamicin, along with 0.1 mg/mL geneticin (G418; Sigma Chemicals, St. Louis, MO, United States).

Techniques: Cell Culture, Lysis, Western Blot, Irradiation, Activity Assay