Adhesive film

Human Bmal1-dLuc U2OS osteosarcoma cells, generated in Dr. Steve A. Kay’s laboratory as previously described [43 (link)], were seeded on 96-well white plates at a density of 3 × 10⁴ cells per well, and incubated in a humidified incubator for 24 h (37 °C, 5% CO₂) in triplicate. In these cells, a lentivirus system (pLenti6; Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) was applied to deliver Bmal1-dLuc reporter genes into U2OS cells and to establish stable reporter lines by using blasticidin as a selection marker.
Exosomes derived from samples taken at time points DS1 or NS5, matched for the time of day (Figure 1), were added for 24 h in the same medium supplemented with depleted FBS (System Biosciences, Mountain View, CA, USA), and then, the cell medium was replaced with 100 µl of luciferase medium and placed in the luminescence module using a Photomultiplier Tube detection system (GloMax-Multi Detection System; Promega, Madison, WI, USA). Plates were sealed with adhesive film (Thermo Fisher Scientific, Waltham, MA, USA). Bioluminescence imaging was performed at 37 °C and recorded every 1 h for 24 h. Total bioluminescence values were normalized to vehicle controls.

Stained bioaerosol samples were arrayed in a custom-designed nanowell device (Edge Embossing), the superstructure of which consisted of two rows of eight wells (16 total) overlaid on an embossed cyclic olefin copolymer (COC) film (Fig 1A and 1B). The COC film contained the 50 x 50 μm nanowells which were arrayed ~140 μm apart center-to-center (Fig 1C). Each microwell therefore comprised approximately 1600 nanowells. Prior to inoculation, the device was plasma coated (Novascan) to counteract hydrophobicity. Following DMN-trehalose staining, the concentrated aerosol sample was added to a single microwell. Samples were loaded and plates sealed using an adhesive film (ThermoFischer Scientific) before centrifuging at 3000 × g for 10 min to disperse the sample for imaging.

All qPCR assays were carried out on a 384-well plate with adhesive film (Thermo Fisher Scientific, Waltham, MA) using a QuantStudio Flex 6 real-time PCR system (Applied Biosystems, Waltham, MA). The reaction mixture was prepared in a final volume of 10 μL, containing 5 μL of 2x PowerUp Sybr Green Master Mix (Thermo Fisher Scientific, Waltham, MA), 2 μL of template DNA, and 3 μL of primer pairs in water at a final concentration of 100 nM, 500 nM, or 900 nM for respective forward and reverse primers as indicated in the text. Reactions with telomere primers and reference gene primers were performed on the same plate. Each assay contained no-template-control (NTC) wells with all reaction components except that water was added instead of template DNA. All reactions were performed in triplicate. The amplification protocol used one of the following four cycling programs (Table 3). All experiments were repeated independently at least three times.
Data acquisition was performed with the QuantStudio real-time PCR software v1.7.2 (Thermo Fisher Scientific, Waltham, MA).