Filter paper

One hundred seeds per treatment were germinated on Filter Paper (Fisherbrand Filter Paper; Cat. No: 09-795B; Quantitative P8; 7.0 cm diameter in size) in a Petri Dish (Kimble Kimax 23060-10020 Borosilicate Glass 100 mm × 20 mm) with 2 mL of either ddH₂O or 75 μg/L AgNPs (in ddH₂O). Seed germination was counted at 10 DAP. Seeds produced by healthy unexposed plants (exposed seed generation 0 or E0) were growing in exposed soil until seed harvest. The seeds from this harvest (E1) were themselves grown on exposed soil until seed harvest to produce the E2 seed generation, and so on for the E3 seed generation. To test reproductive toxicity, germination rates of all 4 generations of seeds (E0–E3) were compared. Each treatment had triplicate germination tests and the entire experiment was repeated three times.
In brief, E0–E3 generations were defined as followed (also see Figure S1):
E0: generation: seeds were harvested from non-exposed plants;
E1: seeds were harvested from the plants germinated from E0 seeds, either irrigated with water or AgNPs;
E2: seeds were harvested from the plants germinated from E1 seeds, either irrigated with water or AgNPs;
E3: seeds were harvested from the plants germinated from E2 seeds. Different letters indicate significantly different.

All substrates were cleaned with acetone and ethanol and dried with nitrogen gas. To one pot solution of 70 wt. % PMMA + SP in acetone, 0 to 50 wt. % of heptadecafluoro−1, 1, 2, 2-tetrahydrodecyl triethoxysilane (FDTES; Gelest) was added. Then, the solution was spray coated on glass and polyester fabric surfaces and dried at ambient conditions. Similarly, one pot solution of PMMA + SP + FDTES was spray coated on different substrates, including acrylic (McMaster), PET (McMaster), aluminum (Fisher), copper (McMaster), tin (McMaster), and filter paper (Fisher) and dried at ambient conditions. Furthermore, one pot solution of 50 wt.% commercially available photochromic pigments (B07123TT3X, Uniglow Pigments) + FDTES and 50 wt. % commercially available thermochromic pigments (B07XB51DL4 and B07D1RNHJF, Atlanta Chemical Engineering) + FDTES were prepared in hexane. Then, the solution was spray coated on polyester fabric and dried at ambient conditions.

Filter paper with a base weight of 103 g/m² and a diameter of 18.5 cm was purchased from Fisher Scientific International, Inc. (Pittsburgh, UK). The paper was made from cellulose fibres with a degree of polymerization (DP) of approximately 830. Sodium hydroxide, urea and zinc oxide were purchased from Nan Jing chemical reagent Co. LTD. (Nan Jing, China). All of the chemicals were used as received.

The procedure was performed as described previously [4]. Briefly, tardigrades were dropped onto a nylon filter (Membrane Solutions, Auburn, WA, USA) placed on a filter paper (Thermo Fisher Scientific, Waltham, MA, USA) in a plastic dish with total of 125 μL water, and then immediately transferred in a sealed plastic box adjusted to 50% or 10% RH and desiccated for 2 days at 18 °C. The desiccated tardigrades were subsequently rehydrated with 2 mL of water and recovery rates at 1 h after rehydration were calculated. Animals showing spontaneous movements or responses to touch stimuli were judged as recovered. Tardigrades desiccated at 10% RH were subjected to 97% RH for 1 day before rehydration with water. Aqueous glycerol solution was used for generating 50% and 97% RH (glycerol; Nacalai Tesque, Kyoto, Japan) [6] and activated silica gel was used for 10% RH. The actual RH was checked using a hygrometer (HN‐CHPR; Chino Corp., Tokyo, Japan).

Freshly isolated ADSCs-Exo in PBS were adsorbed at carbon-coated copper grids (MilliporeSigma, Burlington, USA) for 5 min and the excess was blotted on filter paper (Thermo Fisher Scientific, Waltham, USA). Subsequently, these Exo were stained with 3% uranyl acetate for 5 min and the excess was blotted. The size and morphology of Exo were examined by a transmission electron microscope (Hitachi, Tokyo, Japan). In addition, the size distribution of these Exo was determined by a high-sensitivity nano-flow cytometer (NanoFCM, Fujian, China) as described previously [30 (link)]. Moreover, the profiles of exosomal markers, including Flotillin, CD9 and CD81, and that of Calnexin (1:1000; CST, Boston, USA), an endoplasmic reticulum marker, were detected by western blot. The expressions of CD9 and CD81 (at 1:2 dilution using staining buffer) were further analyzed by flow cytometry.