Settled surface dust was collected using the Omega HEPA Vacuum with a Dust Collection Filter Sock (Midwest Filtration Company, Cincinnati, OH) from 5 different locations within the month of September 2008: floor of the operating facility of a swine confinement facility (housing approximately 400-600 hogs), floor of the feeding barn of a dairy farm (approximately 50 in the building sampled, 2500 total on the facility), floor of the storage facility of a grain elevator (feed grain/feedmill), domestic home without pets (pets never allowed during home ownership), and domestic home with pet, dog (~60 pound dog). House dust was collected in the carpeted living room. In each facility three 1m2 areas were sampled vacuuming the whole area in two orthogonal passes. Dust samples in the Filter Sock were immediately placed in sterile Whirl-Pak bags (Nasco, Ft. Atkinson, WI). Samples were stored at −20°C.
For chemical marker analysis studies, dust samples (5 mg) were extracted with 10ml of 0.05% TWEEN-20 by vortexing for 1 min followed by 1 hr at 100 rpm on a rotary mixer at 20°C. The Tween extraction solution was then aliquoted for separate analysis of endotoxin (rFC), 3-OHFA (GC/MS/MS), muramic acid (GC/MS/MS), and ergosterol (GC/MS). Samples for mass spectrometry were not centrifuged before aliquoting. Individual aliquots were stored at −80°C, then lyophilized prior to sample digestion for mass spectrometry. Although Tween extraction is standard practice for mass spectrometry analysis, Tween is cellular toxic, and thus, not recommended for cell culture studies. All samples were analyzed in triplicate (i.e. three 5 mg extracts were used from each dust sample).
For cell culture studies, the dust samples were placed into solution and sterile filtered by a standard published procedure (Romberger et al. 2002 (link)). Briefly, 100 mg of each dust sample was placed in 1 ml of Hanks’ balanced salt solution (HBSS) without calcium (Biofluids). The mixture was vortexed for 1 min and allowed to stand at room temperature for 1 hour. The mixture was centrifuged at x g for 10 min, and the supernatant was recovered and centrifuged again. The final supernatant was filter (0.22 μm) sterilized. Individual aliquots were stored at −80°C prior to cell culture studies. HBSS extractions were also analyzed by rFC and mass spectrometry.
For chemical marker analysis studies, dust samples (5 mg) were extracted with 10ml of 0.05% TWEEN-20 by vortexing for 1 min followed by 1 hr at 100 rpm on a rotary mixer at 20°C. The Tween extraction solution was then aliquoted for separate analysis of endotoxin (rFC), 3-OHFA (GC/MS/MS), muramic acid (GC/MS/MS), and ergosterol (GC/MS). Samples for mass spectrometry were not centrifuged before aliquoting. Individual aliquots were stored at −80°C, then lyophilized prior to sample digestion for mass spectrometry. Although Tween extraction is standard practice for mass spectrometry analysis, Tween is cellular toxic, and thus, not recommended for cell culture studies. All samples were analyzed in triplicate (i.e. three 5 mg extracts were used from each dust sample).
For cell culture studies, the dust samples were placed into solution and sterile filtered by a standard published procedure (Romberger et al. 2002 (link)). Briefly, 100 mg of each dust sample was placed in 1 ml of Hanks’ balanced salt solution (HBSS) without calcium (Biofluids). The mixture was vortexed for 1 min and allowed to stand at room temperature for 1 hour. The mixture was centrifuged at x g for 10 min, and the supernatant was recovered and centrifuged again. The final supernatant was filter (0.22 μm) sterilized. Individual aliquots were stored at −80°C prior to cell culture studies. HBSS extractions were also analyzed by rFC and mass spectrometry.
