Mco 18aic co2 incubator

HDF cells (ATCC^® PCS20101™) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). HDF cells were cultured in DMEM and F-12 mixed with a ratio of three to one supplemented with 10% heat-inactivated FBS, 100 unit/mL of penicillin and 100 μg/mL of streptomycin. Cells were sub-cultured every 5 days. Cells were incubated at 37 °C under humidified atmosphere containing 5% CO₂ in an incubator (Sanyo MCO-18AIC CO₂ Incubator, Moriguchi, Japan). UVB irradiation was carried out using a UVB meter (UV Lamp, VL-6LM, Vilber Lourmat, France), equipped with a fluorescent bulb emitting 280–320 nm wavelength with a peak at 313 nm. HDF cells were irradiated at a dose of 50 mJ/cm² of UVB in 1 × PBS. Cell medium was subsequently replaced with serum free medium and incubated until analysis.

Sprague–Dawley rats were infected with N. brasiliensis L3 (~3000 larvae) by subcutaneous injection and sacrificed on day seven post-infection [134 ]. We collected faecal pellets on day five and six post-infection. Subsequently, faecal pellets were cultured with activated charcoal—untreated, granular, 8–20 mesh (Sigma-Aldrich, New South Wales, Australia). The culture plates were sealed inside an airtight plastic container in an incubator (Binder, model: BD 115 #02-040007) at 26 °C for one week. After one-week incubation, L3 were harvested, washed with warmed 5× pen/strep PBS, and then transferred to a 12-well culture plate (1500 worms per well) containing warmed 2 mL 5× glutamax, 2× pen/strep PBS media. Plates were incubated in a CO₂ incubator (Sanyo MCO-18AIC CO₂ incubator, SANYO Electric Co., Ltd., Moriguchi, Japan) at 37 °C supplied with 5% CO₂. The supernatant (ESP) was collected and replaced with fresh media twice daily (09:00 and 17:00) for four consecutive days. The supernatants were centrifuged at 3000× g for 30 min, and then aliquot was transferred to Amicon^® Ultra-15, centrifugal 10 kDa filters (Merck Millipore, Victoria, Australia), and centrifuged at 4000× g for 20 min. Concentrated ESP filtrate containing small molecules (<10 kDa) was collected and stored at −80 °C until further analysis.