Nuclepore

The aim of the study was to obtain knowledge about the variation in exposure to fungi during normal indoor activities as a background for variation in concentration and species (examined in part III). To measure short-term variation in exposure, we used the Dekati gravimetric impactor (DGI), a high-volume sampler (Dekati, Finland). The sampler classifies particles into four size fractions with lower cutoff points of aerodynamic diameters (d_ae) of 0.264, 0.608, 1.200, and 2.968 μm when used at a flow rate of 50 liters/min. The particles in the two smallest-size fractions were not used. Airborne particles were sampled on polycarbonate filters (47-mm diameter; pore size, 1 μm; Nuclepore; Whatman) in November 2013. Sampling was performed in 7-min sampling periods before and during the following activities in one or two homes (abbreviated home I and home II): bed making, including shaking of the duvets; cleaning the kitchen; and tidying and vacuum cleaning the living room.

Log-phase P. aeruginosa or S. aureus [200 μl of 10⁸ CFU/ml in saline supplemented with 1% Keratinocyte Basal Medium (Lonza)] were incubated with 0, 45 and 60 μg/ml of KAMP-10 or 0, 60 150 μg/ml of KAMP-18C respectively at 37°C for 3 h. Bacteria were pelleted at 5000 g for 5 min, resuspended in 2.5% glutaraldehyde in PBS and deposited onto a 0.4 μm-pore-size polycarbonate membrane (Whatman Nuclepore) for 1 h at room temperature then 4°C overnight, then post-fixed with 1% osmium tetroxide (OsO₄) for 1 h, 1% thiocarbohydrazide (TCH) for 5 min, and 1% OsO₄ again for 5 min (Willingham and Rutherford, 1984 (link)). After post-fixation, samples were dehydrated with graded ethanol series, then impregnated with 50% hexamethyldisilazane (HMDS) in ethanol, and finally 100% HMDS. The air-dried samples were sputtered with gold before imaging. SEM images were taken with FEI Helios Nanolab 650 at the Swagelok Center for Surface Analysis of Materials (SCSAM) at Case Western Reserve University.

A sterile polycarbonate membrane (PC, Whatman Nuclepore, diameter 2.5 cm, pore diameter 0.2 μm) was carefully placed on a sterile TSA plate and inoculated at its center with 50 μL of an overnight TSB culture of E. coli/GFP-E. coli grown at 37°C in aerobic/anaerobic conditions. Inocula were normalized through optical density (OD) measurements at 600 nm with a JENWAY 7315 Spectrophotometer, to obtain a final concentration of 10⁸ cells/mL. The membrane was left on the agar plate until the inoculum had dried completely, after which it was carefully transferred to inside the transwell (ThinCert^TM Cell Culture Inserts with translucent PET membrane – Greiner bio-one), inlaid in a six well culture plate (Greiner bio-one). One mL of TSB medium (pre-reduced if working in anaerobic conditions) was added to the plate well (basolateral compartment). Biofilm was grown at 37°C in both aerobic and anaerobic conditions, and to guarantee continuous growth, the transwells were transferred every 24 h to new plate wells with fresh TSB. Basolateral media were collected every 24 h and stored at -80°C to be used in further experiments with Caco-2 cells (aerobic part of the model).

Particulate carbon was collected on pre-combusted GF/F filters (25 mm, Whatman Nuclepore), which were stored at -20°C until analysis. The filtration volume (80–380 mL) was adjusted to the particle loading of the seawater. Filters were treated with 200 μL of carbon-free HCl (0.2 N) to remove inorganic carbon and dried at 60°C for approximately 48 h. After drying, filters were packed into small Zn-cartridges and analyzed using an elemental analyzer (Euro EA 3000, EuroVector Instruments & Software, Italy).
For analysis of DOC concentration, seawater was filtered through combusted (400°C, 4 h) GF/F filters (0.7 μm, Whatman), and 15 mL was sealed in combusted glass ampoules. Samples were stored at -80°C in the dark. Some of the ampoules were damaged during transport to the mainland, resulting in reduced temporal resolution. Acidified samples (pH 2, HCl) were analyzed using high-temperature catalytic oxidation (Sugimura and Suzuki, 1988 (link)) on a Shimadzu total organic carbon (TOC)-VCPH instrument with analytical precision better than 5% for three replicate samples. Accuracy was tested in each run against deep Atlantic seawater reference material (D.A. Hansell, University of Miami, FL, USA) and was better than 5%.
Total organic carbon (TOC) was calculated as the sum of particulate organic carbon (POC) and DOC.

For total cell counts, formaldehyde-fixed water samples were directly concentrated on filters. The cells on the filters were stained with SYBR Green I (Invitrogen) and embedded with moviol on black polycarbonate membrane filters (0.2 µm, Nuclepore, Whatman)^{104 (link)}. The stained cells on the filters were counted by epifluorescence microscopy. Total cell counts were determined for the two open ocean seawater, plume and hydrothermal fluid samples.

Liposomal doxorubicin (L-Dox) was prepared as previously reported [27 (link)]. Briefly, L-Dox was prepared with HSPC/cholesterol/DSPE-PEG at a molar ratio of 5.5/4/0.5, respectively, using thin lipid film hydration in ammonium sulfate (250 mM), followed by heated extrusion [28 (link)]. Small unilamellar vesicles were obtained by extrusion through Nuclepore^® (Whatman Inc., Clifton, NJ, USA) filters with pore sizes ranging from 200 to 100 nm on a Thermo barrel extruder at 65 °C. Doxorubicin was loaded based on an ion gradient method called remote loading [28 (link),29 (link)]. The diameter and polydispersity index (PDI) were measured using a Zetasizer nano-ZS (Malvern Instruments Ltd., Malvern, UK). L-Dox with size of approximately 100 nm and a PDI below 0.1 was used in this study.

Matrigel (reconstituted basement membrane; 25μg) was dried on a polycarbonated filter (PVP free, Nuclepore, Whatman, Maidstone, UK). Fibroblast conditioned medium (obtained from confluent NIH-3T3 cells cultured in serum free DMEM) was used as the chemoattractant. Cells were harvested by brief exposure to 1mM EDTA, washed with DMEM containing 0.1% bovine serum albumin and added to the Boyden chamber (200,000 cells). Cells were incubated for 6h at 37°C in humidified atmosphere of 95% air and 5% CO₂. Cells that traversed the Matrigel layer and attached to the lower surface of the filter were stained with Diff Quik kit (Dade Diagnostics, Aguada PR) and counted in five randomized fields. The mean of the counts was calculated for each cell line and are expressed as the mean ± SE.