Exposure to the antimicrobial chemical triclosan disrupts keratinocyte function and skin integrity in a model of reconstructed human epidermis

Data Collection Methods

1. Tissues
   1. EpiDerm (MatTek) tissues were incubated in 0.9 mL or 5 mL media/well
2. Triclosan Exposures
   1. Tissues were exposed on the apical side to acetone or triclosan (0.05-0.2%) once for 6, 24, or 48 hours or once/day for 5 days
   2. Tissues were incubated at 37 °C with 5% CO₂ during the exposure
3. LDH Release Assay
   1. The LDH-Cytotoxicity Colorimetric Assay Kit II (BioVision) was used
   2. Absorbance (450 nm) was measured using a microplate spectrophotometer in duplicate for each sample
4. Gene Expression Analysis
   1. Tissues were washed with 100 µL DPBS immediately prior to tissue collection
   2. Tissues were disrupted and homogenized in 700 µL QIAzol using a steel bead and TissueLyser II or with a pellet pestle homogenizer
   3. Total RNA was isolated from lysates using the miRNAeasy kit (Qiagen)
   4. RNA purity and yield were determined on a NanoDrop Spectrophotometer
   5. Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
   6. TaqMan Fast Universal PCR Master Mix (Applied Biosystems), cDNA, and gene-specific primers (TaqMan Gene Expression Assays) were combined and real-time quantitative PCR was performed
   7. Genes evaluated include FLG, FLG2, IVL, LOR, TJP1, OCLN, KRT10, KRT14, CDH1, TSLP, S100A8, IL-1A, IL-1B, TNF, CXCL1, CXCL2, CXCL8
5. Cytokine Release
   1. Evaluated with a custom human premixed multi-analyte kit (LXSAHM; R&D Systems) and analyzed on a MagPix (Luminex)
   2. Analytes measured: EGF, IL-1A, IL-6, IL-18, IL-33, S100A8, TNF-A, VEGF, GM-CSF, IL-1B, IL-8, IL-31, IL-36B, TGF-A, TSLP
6. Permeability Assay
   1. Tissues were washed with 100 µL DPBS immediately prior to assay
   2. Tissues were exposed on the apical side to Lucifer Yellow CH dilithium salt (Sigma) and incubated at 37 °C with 5% CO₂ for 2 hours
   3. Media was collected and fluorescence intensity was measured in triplicate
   4. Read on a microplate reader (excitation 428 nm, emission 536 nm)
7. Histology
   1. Formalin-fixed paraffin-embedded tissues (5 µm) were mounted on slides and stained with hematoxylin and eosin
   2. Slides were brightfield imaged and epidermal thickness was measured