Collagen 1 from rat tail

Freshly isolated PBMCs were resuspended in complete RPMI 1640 (Gibco) supplemented with 10% autologous human plasma, 1 mM sodium pyruvate (HyClone, Logan, UT, USA), 25 mM hepes, 100 μg/mL penicillin/streptomycin (HyClone), 2mM L-glutamine, and 1X non-essential amino acids. In a 24-well plate coated with collagen I from rat tail (Corning, Corning, NY, USA), 2.5 × 10⁶ cells/well in 450 μL were seeded in triplicates and rested overnight at 37 °C in a 5% CO₂ incubator. To replicate the inflammatory milieu of AD skin, PBMCs were stimulated with 2 ng/mL of staphylococcal enterotoxin B (SEB) (Toxin Technologies, Sarasota, FL, USA) and 120 ng/mL of recombinant human thymic stromal lymphopoietin (TSLP) (Biolegend, San Diego, CA, USA) at Day 0. Every 2 days, complete RPMI was added to each well. On day 7, the cells were harvested and cell count for each condition was performed. In 96-well V bottom plates, 2.5 × 10⁵ cells/well were plated for subsequent flow cytometry experiments.

Substrates were made on 35 mm glass bottom imaging dishes which were treated for 5 minutes with UV-ozone. 1% v/v of 3-aminopropyltriethoxysilane (APTES) in deionized water were added and allowed to sit for 25 minutes at room temperature. The dishes were washed thoroughly with deionized water and brought to the biosafety hood for sterile handling. 1 mL of sterilized MilliQ water used to rinse the dish before collagen was added.
For collagen preparation, microcentrifuge tubes and reagents used were kept on ice for as long as possible while handling. Collagen I from rat tail (Corning, Corning, NY) was diluted with deionized water such that the final concentration was either 1.2 mg/mL or 3.0 mg/mL. 100 µL of 10X phosphate buffer saline (Thermofisher Scientific, USA) containing phenol red was added dropwise while vortexing as a pH indicator. 0.5 N NaOH was then added dropwise to the mixture with periodic vortexing until the solution became a slight pink (pH~7). The collagen was then added to the treated imaging dish (total volume of 1 mL) and incubated at 20 °C for 1 hour and then at 37 °C, 5% CO₂ overnight. 0.25 × 10⁶ cells were then added to each dish the next day and then allowed to incubate at 37 °C, 5% CO₂ overnight again before imaging took place.

Cells were trypsinized and 10⁴ cells/ml were re-suspended in RPMI medium containing 10% FBS and 1% Penicillin-Streptomycin (Life Technologies). Then 100 µl of cell suspension was plated in 48-well plates coated with 1% agarose (Life Technologies) and incubated for 3 days. In each well, a spheroid was formed from 10³ cells. Next, the spheroids were plated on Lab-Tek chambers (Sigma), in a mixture of collagen I from rat tail (Corning) at a final concentration of 2 mg/ml, PBS, sodium hydroxide (NaOH) and serum-free medium. The spheroids were monitored for 5 consecutive days by using an inverted Leica microscope (Wetzlar, Alemanha) equipped with camera device using 4x objective.