Novel Combination Of Antioxidants To Restore Healthy Intercellular Communication In An In vitro Model Using Conditioned Medium From Senescent Cells

Summary

This method studies how senescent skin fibroblasts affect healthy ones. Senescence is induced via UVB exposure, and the conditioned medium is applied to normal fibroblasts. It helps evaluate bioactive compounds targeting altered cell-to-cell communication in skin aging.

Abstract

Skin aging is a multifactorial process driven by alterations at the molecular and cellular level, also known as hallmarks of aging. One of these alterations consists of altered intercellular communication, which contributes to skin tissue dysfunction and structural damage. Here, an in vitro model to mimic these skin aging features by incubating healthy fibroblasts with conditioned medium from senescent fibroblasts is presented. First, UVB-irradiated fibroblasts can be analyzed for changes in gene expression associated with photoaging. Second, conditioned medium from UVB-induced senescent fibroblasts can be harvested and transferred to healthy fibroblasts, leading to downregulation of extracellular matrix proteins and upregulation of proinflammatory cytokine gene expression. These changes are counteracted by using a novel combination of antioxidants (vitamin C + idebenone). These results demonstrate that the in vitro model can be used to assess the efficacy of novel compounds on skin dermal cell aging features, and thus to select compounds that may modulate skin regenerative and rejuvenation cellular processes.

Introduction

Skin aging is the natural process of decreased skin functions and properties, including loss of firmness and elasticity and increased sagging. This is accelerated by external factors, such as ultraviolet (UV) exposure and pollution, which damage cells and cause changes at the molecular level¹. These changes have been well characterized by recent research and identified as hallmarks of aging, including genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, defective macroautophagy, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis². These features explain how subcellular damage translates into tissue dysfunction, thereby favoring the aging process. Interestingly, these alterations do not act independently but rather form an interdependent network signature. Primary damage, such as genomic damage or epigenetic alterations, accumulates and eventually causes cellular senescence. In the case of the skin, this is mainly caused by UV damage, with photoaging being the main contributor to skin aging traits³. Senescent cells show increased production of proinflammatory cytokines, matrix metalloproteinases, and growth factors, which is also known as senescence-associated secretory phenotype (SASP) and includes IL6, IL8, MMP1, MMP3, TNFA, or IL1A, among others^4,5,6. These secreted factors not only affect the structural properties of the skin, such as the dermal extracellular matrix, but also disrupt the function of surrounding cells. This includes the release of cytokines that stimulate melanocyte activation to provoke age-associated skin hyperpigmentation (such as Stem Cell Factor or IL18) or other damaging proteins that alter the function of surrounding healthy fibroblasts, compromising their function and contributing to the enhanced amplification of the senescent phenotype^7,8,9,10,11. As a result, chronic inflammation and altered intercellular communication are key promoters of the damaging state that translates these molecular alterations to visual signs of skin aging, such as wrinkles, sagging, age spots, or loss of firmness.

The specific protein contributors to the detrimental effects of SASP can be studied through well-known techniques such as enzyme-linked immunosorbent assay (ELISA), Luminex, quantitative real-time polymerase chain reaction (qRT-PCR), or Western Blot, among others^5,12. However, this approach is useful for identifying specific proteins from an individual perspective and characterizing a specific mechanism of action, but it does not consider the effects of the full SASP on neighboring cells. Here, an in vitro protocol is proposed to study the effect of the SASP produced by UVB-induced senescent dermal fibroblasts on healthy neighboring fibroblasts, mimicking the altered intercellular communication hallmark of aging. As stated before, UV is the main driver of cell senescence in the skin. Hence, UV damage is selected as a source of damage over replicative or oncogene-induced senescence to build the current model³. For this, the medium containing the factors secreted by senescent fibroblasts (conditioned medium) is used, and markers of cell function are quantified in healthy fibroblasts incubated with this medium. Under this protocol, once the conditioned medium has been transferred from senescent to healthy cells, UVB-induced senescent fibroblasts are harvested to quantify molecular features, such as genes involved in proinflammatory and paracrine signaling. Secondly, healthy fibroblasts treated with conditioned medium from senescent fibroblasts are harvested to characterize the SASP's effect on neighboring cells, quantifying genes involved in extracellular matrix, growth factors, and fibroblast activation. Thus, the effect of compounds of interest (COI) is analyzed both initially in senescent fibroblasts and subsequently in healthy fibroblasts treated with the conditioned medium. Regarding these compounds, previous research has shown that antioxidants are effective in protecting against and correcting signs of photoaging-induced skin aging. Consequently, the combination of two antioxidants (vitamin C and idebenone) is used to revert some of the alterations induced by UVB, both in senescent cells and healthy cells incubated with conditioned medium from senescent cells^13,14,15

Overall, this protocol is designed to study the effects of SASP on skin cells and to identify novel molecules/interventions that restore altered intercellular communication, thereby being appropriate for regenerative and bio-stimulating products in aesthetic medicine and regenerative dermatology.

Protocol

Human dermal fibroblasts used in this study were obtained from a commercial cell line provider. As no primary human samples, patient data, or in vivo experiments were involved, Institutional Review Board (IRB) approval and informed consent were not required for this study.

1. Senescence induction in dermal fibroblasts using UVB

1. Plate human dermal fibroblasts at a density of 6 × 10⁴ cells per well in a 6-well plate using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS).
2. After 24 h, remove the cell culture medium and replace it with 1 mL of phosphate buffered saline (PBS) per well.
3. Irradiate the cells with UVB light at 25 mJ/cm².
   NOTE: This energy has been optimized for the UVB irradiation device used in this protocol (see Table of Materials) to induce sufficient damage to trigger senescence while avoiding excessive damage that may lead to apoptosis. When using other devices, adjust the irradiation energy accordingly.
   CAUTION: UVB radiation is hazardous. Use appropriate protective equipment and shielding to avoid exposure.
4. Remove PBS and add 2 mL of fresh cell culture medium per well.
5. After 72 h, remove the cell culture medium, add 1 mL of PBS per well, and repeat UVB irradiation as described in step 1.3.
6. After 72 h, remove the cell culture medium and add 0.5 mL of 0.05% trypsin-EDTA per well. Incubate for 1–2 min at 37 °C, then harvest the cells using 1.5 mL of fresh cell culture medium.
7. Plate the cells at a density of 8 × 10⁴ cells per well in a 6-well plate using cell culture medium (DMEM supplemented with 10% FBS). Cellular senescence is induced after this step.

2. Treatment of senescent cells with compounds of interest

1. After 24 h, remove the cell culture medium and add 2 mL of cell culture medium containing compounds of interest.
2. After an additional 24 h, plate a separate batch of human dermal fibroblasts (as described in step 1.1) in 6-well plates. These cells will serve as healthy dermal fibroblasts for the transfer of conditioned medium.

3. Conditioned medium transfer from senescent cells to healthy cells

1. At 48 h after compound treatment of senescent cells and 24 h after plating healthy fibroblasts, collect 2 mL of conditioned medium per well from senescent cells and transfer it to the healthy fibroblasts.
   NOTE: Remove the culture medium from healthy fibroblasts immediately before adding the conditioned medium to ensure efficient transfer.
2. Add 1 mL of PBS to the senescent cells and harvest them using trypsin as described in step 1.6. Use these cells for analysis of senescence or inflammation markers by using an ELISA kit or qPCR.
   NOTE: Cell pellets may be stored at −80 °C if downstream assays are not performed immediately.
3. Incubate healthy fibroblasts receiving conditioned medium at 37 °C with 5% CO₂ for 24 h.

4. Healthy cells harvest for fibroblast activation analysis by gene expression analysis

1. After 24 h incubation with conditioned medium, harvest healthy fibroblasts using trypsin as described in step 1.6. Analyze extracellular matrix gene expression by qPCR.
2. Perform qPCR analysis.
   1. Extract RNA from cell pellets according to the manufacturer’s instructions (RNA extraction kit).
   2. Perform reverse transcription of 200 ng RNA according to the manufacturer’s instructions (Reverse transcription kit).
   3. Amplify cDNA according to the manufacturer’s instructions (qPCR master mix). Perform qPCR under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Primers used are listed in Table 1.

Gene	Forward sequence	Reverse sequence
CDKN2A	5’-GGCATTGTGAGCAACCACTG-3’	5’-CCTGTAGGACCTTCGGTGAC-3’
CDKN1A	5'-CTGGAGACTCTCAGGGTCGAA-3'	5'-CCAGGACTGCAGGCTTCCT-3'
SCF	5'-AATCCTCTCGTCAAAACTGAAGG-3'	5'-CCATCTCGCTTATCCAACAATGA-3'
IL-18	5'-TGCAGTCTACACAGCTTCGG-3'	5'-GTTTGTTGCGAGAGGAAGCG-3'
ELN	5’-GCAGGAGTTAAGCCCAAGG-3'	5’-TGTAGGGCAGTCCATAGCCA-3'
FBN1	5’-GGTGAATGTACAAACACAGTCAGCAG-3'	5’-ATAGGAACAGAGCACAGCTTGTTGA-3'
IL-6	5’-AGCCCTGAGAAAGGAGACATGTA-3'	5’-TCTGCCAGTGCCTCTTTGC-3'
IL-8	5’-ATTTCTGCAGCTCTGTGTGAAGGT-3'	5’-TTTTTTATGAATTCTCAGCCCTCT-3'
CTGF	5’-GGAAATGCTGTGAGGAGTGGGTGT-3´	5´-TGTCTTCCAGTCGGTAGGCAGCTA-3
IGF1	5’-CAGAGCAGATAGAGCCTGCG-3'	5’-CAGGTAACTCGTGCAGAGCA-3'

Table 1: List of primers used for qPCR assays. Forward and reverse sequences are provided for each primer corresponding to the genes studied and quantified in this model.

Results

The present protocol describes a method to simulate the altered intercellular communication hallmark of aging in vitro. Figure 1 presents a graphical schematic illustrating the main steps of the protocol, including senescence induction, marker quantification, and the analysis of altered intercellular communication in skin cells in vitro.

Figure 1: Graphical summary of the protocol, including the main steps. A schematic summary of the main steps of the protocol. First, senescence is induced using UVB damage in healthy fibroblasts. Next, senescent fibroblasts are treated with the compound of interest (COI), and cells are harvested to assess SASP and epigenetic alterations. In parallel, conditioned medium is transferred to healthy fibroblasts to characterize the effect of secreted factors on extracellular matrix and inflammation markers in neighboring healthy cells. Please click here to view a larger version of this figure.

Human dermal fibroblasts are initially induced into senescence by repeated UVB irradiation, thereby simulating the process of skin photoaging. Once induced, these senescent fibroblasts exhibit characteristic senescent features (Figure 2), produce proinflammatory cytokines (Figure 3), and display altered epigenetic modifications associated with aging (Figure 4), confirming the senescent phenotype.

Figure 2: Validation of positive senescence markers in UVB-irradiated fibroblasts. (A, B) Beta-galactosidase-associated senescence staining (10× magnification) of non-irradiated control cells (A) and UVB-irradiated control cells (B). (C) Gene expression of canonical senescence cell cycle regulators (CDKN2A and CDKN1A). Error bars represent standard deviation (SD, n = 3). Statistical analysis was performed using Student’s t-test, where “a” indicates p < 0.05 comparing control vs UVB control, and “b” indicates p < 0.01 comparing control vs UVB control. Please click here to view a larger version of this figure.

Figure 3: Quantification of proinflammatory gene expression in senescent fibroblasts. Gene expression levels of SCF and IL18 in control cells, UVB-induced senescent fibroblasts, and UVB-induced senescent fibroblasts treated with idebenone (1 µM) and vitamin C (3 mM) for 48 h. Error bars represent standard deviation (SD, n = 3). Statistical analysis was performed using Student’s t-test, where “a” indicates p < 0.01 comparing control vs UVB control, “b” indicates p < 0.05 comparing control vs UVB control, and “c” indicates p < 0.001 comparing UVB control vs UVB treated with the compounds. Please click here to view a larger version of this figure.

Figure 4: Quantification of age-associated epigenetic alteration in senescent fibroblasts. H3K27me3 methylation levels in UVB-induced senescent fibroblasts and UVB-induced senescent fibroblasts treated with idebenone (1 µM) and vitamin C (3 mM) for 48 h, compared to non-senescent control cells. Error bars represent standard deviation (SD, n = 3). Statistical analysis was performed using Student’s t-test, where “a” indicates p < 0.05 comparing control vs UVB control, and “b” indicates p < 0.05 comparing UVB control vs UVB treated with compounds. Please click here to view a larger version of this figure.

As shown in Figure 3, the photoaging-associated cytokines stem cell factor (SCF) and IL18 are upregulated in senescent dermal fibroblasts. In addition, senescent fibroblasts exhibit altered levels of the epigenetic marker H3K27me3 (Figure 4). In both cases, these alterations are reversed by treatment with a combination of antioxidants (vitamin C and idebenone). This demonstrates the utility of the method not only for modeling the aging process in vitro but also for screening potential compounds that may target this process and mitigate the detrimental effects of senescent cells.

These detrimental effects of senescent cells are further observed when conditioned medium from senescent cells is applied to healthy fibroblasts. As shown in Table 2, the expression of extracellular matrix proteins (ELN, FBN1), growth factors (CTGF, IGF1), and proinflammatory cytokines (IL6, IL8) in healthy fibroblasts is dysregulated after 24 h of incubation with conditioned medium. Notably, treatment with a combination of vitamin C and idebenone restores these changes by upregulating extracellular matrix proteins and growth factors while downregulating proinflammatory cytokines (Table 2).

	Normal fresh growth medium	Conditioned medium  from senescent cells
Gene	Control	Control	Vitamin C + idebenone
ELN	1.00	0.68 (±0.07) a	0.87 (±0.05) c
FBN1	1.00	0.80 (±0.01) b	0.93 (±0.07) c
IL-6	1.00	1.53 (±0.13) a	1.13 (±0.14) c
IL-8	1.00	1.07 (±0.21)	0.41 (±0.24) c
CTGF	1.00	0.94 (±0.07)	1.33 (±0.10) c
IGF1	1.00	0.49 (±0.06) b	5.55 (±0.73) d

Table 2: Effect of conditioned medium from senescent fibroblasts on healthy fibroblast function. Gene expression of extracellular matrix (ELN, FBN1), inflammation (IL6, IL8), and growth factor (CTGF, IGF1) markers in healthy fibroblasts incubated for 24 h with conditioned medium from senescent fibroblasts treated or not with idebenone (1 µM) and vitamin C (3 mM) for 48 h. Standard deviation (SD, n = 3) for each quantification is provided in brackets, and statistical analysis was performed using Student’s t-test, where “a” indicates p < 0.05 comparing non-treated control vs conditioned medium-treated control; “b” indicates p < 0.01 comparing non-treated control vs conditioned medium-treated control; “c” indicates p < 0.05 comparing conditioned medium-treated control vs conditioned medium-treated control exposed to idebenone + vitamin C; and “d” indicates p < 0.001 comparing conditioned medium-treated control vs conditioned medium-treated control exposed to idebenone + vitamin C.

Discussion

Understanding the aging process at the molecular level is key to designing novel approaches and interventions that delay aging-related features and prolong the longevity and functionality of cells and tissues. The definition and identification of the hallmarks of aging have been essential in establishing a scientific framework for the development of these interventions^2,16. Among the recognized hallmarks of aging, altered intercellular communication constitutes a fundamental feature, characterized by compromised and dysfunctional signaling between cells that precipitates age-associated tissue degeneration. In aged skin, the accumulation of senescent cells promotes a proinflammatory microenvironment that alters the functional capacity of adjacent non-senescent cells, which, in turn, exacerbates tissue damage by diminishing cellular activity. This phenomenon culminates in reduced synthesis of extracellular matrix proteins and growth factors, along with increased levels of deleterious proteins such as matrix metalloproteinases, ultimately manifesting as visible signs of skin aging, including dull complexion, reduced firmness, and the emergence of wrinkles^6,17,18.

Given the complexity of fully characterizing the cell secretome, a novel protocol is proposed to study the global paracrine effects of secreted factors from senescent dermal fibroblasts on healthy fibroblasts. First, senescence is induced in human dermal fibroblasts using UVB light, as UV damage is one of the primary drivers of skin photoaging¹³. Senescence induction is a critical step in this protocol, as cells must acquire the full senescence-associated secretory phenotype (SASP) to simulate the altered intercellular communication observed during aging. The senescent state is established after cells experience chronic, irreparable damage, leading to a permanent cessation of cell proliferation as a protective mechanism to prevent the propagation of damaged cells. The level of damage must be carefully modulated to induce senescence without causing excessive damage that could lead to cell death (apoptosis). As described in the protocol section, UVB energy must be optimized in advance to ensure proper senescence induction. In this protocol, two UVB irradiations separated by several days are sufficient to achieve this state, whereas a single high-dose exposure leads to apoptosis rather than senescence. The upregulation of CDKN2A (p16) and CDKN1A (p21) gene expression, along with positive beta-galactosidase staining, confirms the successful induction of the senescent phenotype.

Once cells are exposed to the senescence-induction protocol, markers are quantified to confirm its success. On one hand, UVB-irradiated fibroblasts exhibit an increase in H3K27me3 (Figure 4), which correlates with epigenetic alterations in damaged cells^19,20. Previous research has shown that this epigenetic modification is involved in regulating cellular damage and repair processes in senescent cells. An increase in this marker correlates with repression of longevity-associated genes and the formation of senescence-associated heterochromatin foci, consequently representing a form of epigenetic drift associated with aging hallmarks^21,22. Thus, it is currently employed as a marker of epigenetic aging alterations.

On the other hand, these fibroblasts also show upregulation of classical senescence markers, including senescence-associated beta-galactosidase staining and increased expression of CDKN2A (p16INK4a) and CDKN1A (p21WAF1/CIP1) (Figure 2)²³. Interestingly, these cells also show upregulation of additional proinflammatory cytokines that contribute to photoaging and age-associated hyperpigmentation, including SCF and IL18 (Figure 3)^7,8,9,10,11. Notably, these cytokines can stimulate melanogenesis in epidermal melanocytes, highlighting the utility of this model for screening compounds that modulate dermal influence on skin pigmentation in the context of photoaging⁷. These results confirm the successful induction of the senescent state. As shown in Figure 3 and Figure 4, the combination of compounds used in this protocol (vitamin C + idebenone) restores changes in H3K27me3 levels and SCF and IL18 gene expression, indicating the potential of this model to screen for compounds that regulate proinflammatory cytokine expression and epigenetic alterations in senescent fibroblasts. The effect of these antioxidants on classical senescence markers is not evaluated in the current protocol, but could be included as a complementary step.

The next step in the protocol involves transferring conditioned medium from senescent fibroblasts (which secrete factors) to healthy fibroblasts to characterize the effect of SASP on neighboring cells, reflecting the functional consequences of altered intercellular communication. This approach enables quantification of how fibroblast function markers, including extracellular matrix components, growth factors, and inflammatory cytokine gene expression, are affected by paracrine factors released from senescent cells. Nonetheless, the potential effect of residual compounds in the conditioned medium should not be underestimated, and thus, the observed results may be influenced by both paracrine factors produced by senescent cells and residual concentrations of compounds used during the treatment step. A critical aspect of this protocol is the transfer of conditioned medium from senescent to healthy cells on the same day. In contrast, other studies freeze conditioned medium and thaw it when healthy cells are not immediately available. Although secreted factors are expected to remain stable when frozen at −80 °C, this has not been validated in the present model; therefore, the potential impact of freeze–thaw cycles remains to be determined.

As expected, exposure of healthy fibroblasts to conditioned medium from senescent fibroblasts alters the expression of extracellular matrix components, growth factors, and inflammatory cytokines (Table 2). Notably, treatment with vitamin C plus idebenone reverses these changes, demonstrating that the model effectively represents altered intercellular communication in aged cells and supporting the potential of this combination as a promising approach for dermatological and medical aesthetic applications aimed at mitigating aging features and promoting skin cell longevity.

Regarding alternative approaches, previous studies have employed antibody-based techniques such as ELISA, Western blot, or Luminex^24,25,26. These methods are useful for identifying and characterizing specific proteins within the SASP; however, they do not capture the effects of the complex mixture of proteins and factors present in the secretome. In contrast, the use of conditioned medium from senescent cells—containing a diverse array of secreted biological molecules—provides a more comprehensive approach to evaluating altered intercellular communication in vitro. Previous studies have used conditioned medium transfer to assess the effects of secreted factors under defined conditions^27,28, including its impact on other cell types such as stem cells or keratinocytes^29,30. In this protocol, a method is proposed to characterize the paracrine effects of senescent dermal fibroblasts on neighboring healthy fibroblasts, identifying a specific gene expression signature involving extracellular matrix, growth factor, and inflammatory pathways. This approach represents a valuable in vitro tool to model altered intercellular communication associated with aging.

Limitations

A primary limitation of the present study lies in the lack of identification of the specific mechanisms or proteins responsible for the observed alterations. In the absence of proteomic techniques, this protocol evaluates the global effect of the SASP without addressing the specific contribution of cytokines, growth factors, or other proteins secreted by senescent fibroblasts.

A further limitation concerns the origin of the conditioned medium, which is obtained from cells previously treated with the tested compounds. As a result, residual amounts of these compounds may remain after treatment of healthy fibroblasts. Thus, the observed effects on healthy fibroblasts may be attributable not only to secreted factors from senescent cells but also to residual compounds initially supplied during the treatment of senescent cells. To address this issue, an improved experimental design could involve treating senescent cells with the compounds for 48 h, followed by replacement with fresh cell culture medium and an additional 24 h incubation, after which the medium is harvested and transferred to healthy fibroblasts.

Another relevant consideration is the potential influence of cell number on the observed outcomes. Although a defined number of cells is initially seeded, the treatment may alter the final cell count and, consequently, affect the observed results in healthy fibroblasts. Therefore, harvested cells could be counted, allowing the volume of conditioned medium to be adjusted before transfer. In addition, the current protocol focuses on evaluating the impact of compounds on proinflammatory cytokines in senescent fibroblasts, but does not address their effects on classical senescence markers such as p16, p21, or beta-galactosidase staining.

Finally, it should be noted that conditioned medium derived from cultured senescent fibroblasts may not fully recapitulate the complexity of in vivo skin aging or systemic inflammatory signaling processes.

Troubleshooting

Following UVB irradiation, fibroblasts may show unaltered levels of H3K27me3, SCF, and IL18 gene expression compared to control cells, indicating that senescence has not been successfully induced; in this case, the UVB dose should be checked and adjusted. Conversely, if cells exhibit a high level of apoptosis, reflected by a significant reduction in viable cell number, this suggests that excessive damage has been produced, and the UVB energy should be reduced.

Possible optional modifications of the protocol

Possible optional modifications of the protocol (not validated within the current study) include expanding the biomarker readout to evaluate additional markers in both senescent and healthy fibroblasts, particularly those involved in pathways not considered here, such as mitochondrial function or metabolism. Furthermore, the senescent state may be induced using alternative stimuli, including replicative senescence achieved through repeated passaging until proliferation ceases. In addition, conditioned medium from senescent fibroblasts may be applied to other skin cell types, such as keratinocytes or melanocytes, to investigate additional biological processes, including pigmentation, epidermal regeneration, or barrier function.

Conclusion

In conclusion, this protocol demonstrates the utility of the method as a screening tool for identifying novel active ingredients or interventions that regulate altered intercellular communication in the dermis, a key molecular hallmark of aging. This research provides a model that represents the influence of senescent fibroblasts on healthy fibroblasts in the context of skin photoaging, enabling the study and evaluation of both senescent and non-senescent cellular states. As a positive control, the proposed protocol highlights the potential of a vitamin C and idebenone combination as an effective antioxidant cocktail with regenerative and reparative properties in aged human dermis.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well plates	Fisher Scientific	10578911	Cell culture multiwell plates to use during the assay (senescence induction and compound treatment)
Bio-LINK Crosslinker BLX-312/36 (UVB irradiation device)	Vilber Lourmat	VL-6111-1090-1	Device used to produce damage by UVB irradiation and consequently induce senescence
Dulbecco's Modified Eagle Medium (DMEM)	Merck Life science	D6046-500ML	Standard cell culture medium for dermal fibroblasts
Fetal bovine serum (FBS)	Fisher Scientific	17593595	Nutrient supplement used a a source of growth factors, hormones, proteins and essential micronutrients for cell growth
H3K27me3 ELISA kit	Epigentek	P-3014T-096	A specific immunoassay to detect the epigenetic modification of H3K27me3 in biological simples
Human dermal fibroblasts	Promocell	C-12302	Main skin cell in the dermis and regulator of skin photoaging
Phosphate buffered saline (PBS)	Merck Life science	D8537-500ML	Buffer solution that mimicks physiological environment. Used to wash cells without inducing strss or reducing cell viability
PrimeScript RT reagent kit (reverse transcription kit)	Takara Bio	RR037A	The kit to obtain cDNA from RNA isolated from cells. 2nd step for qPCR assay after RNA purification.
TB Green Premix Ex Taq  (qPCR master mix)	Takara Bio	RR420A	The kit to produce the polymerase chain reaction from cDNA to quantify gene expression levels. 3rd step for qPCR assay after reverse transcription.
Total RNA Purification Kit (RNA extraction kit)	Norgen	17200	The kit to obtain RNA from harvested cells. 1st step for qPCR assay after cell harvesting.
Trypsin/EDTA 0.05%	Merck Life science	T2601-100ML	Enzyme solution used in harvesting/passaging steps for cell detachment from cell culture plates