Rpmi 1640

Human thymuses from newborns and young children were used as a source of thymocytes. Mouse thymuses were obtained from 4–6 week old C57BL/6J mice. Human and mouse thymuses were crushed through 70 µm nylon cell strainer (BD Falcon) and thymocytes were collected in RPMI 1640 (Wisent). Before seeding, human and mouse thymocytes were incubated in presence of 1µM cell tracker CMFDA in RPMI without FBS for 15 minutes in the dark and washed twice with PBS 1X. Human and mouse thymocytes were seeded at 5.10⁶ cells/ml in 12 ml of RPMI 1640 (Wisent) with 5% FBS in 25 cm² flasks (BD Falcon). After 24 hours, supernatants were collected and centrifuged 10 minutes at 800g to eliminate cells, debris and larger apoptotic bodies. Supernatant containing MPs were then collected, aliquoted and kept at -80°c.

Expression of the galactosaminogalactan (GAG) cluster genes AFUA_3G07910 (uge3), AFUA_3G07900 (sph3), AFUA_3G07890 (ega3), and AFUA_3G07860 (gtb3) in the Δagd3 mutant was compared to the wild-type Af293. The fungal strains indicated in the figures were grown for 18 h at 37°C under various growth conditions including phenol-free RPMI 1640 (Wisent) buffered with morpholinepropanesulfonic acid (MOPS) (Bioshop, Inc.) at pH 5.4 and pH 7.0, Brian medium (3 (link)), or Aspergillus medium (2 (link)) with iron supplementation of 0, 2, or 30 mM. For anaerobic (AnaeroPack; Mitsubishi Gas Chemical, Inc.) or microaerophilic (MicroAeroPack; Mitsubishi Gas Chemical, Inc.) conditions, fungi were grown in phenol-free RPMI 1640 (Wisent) buffered with MOPS (Bioshop, Inc.) at pH 7.0 in anaerobic chambers with the appropriate gas packs. Mycelia were collected, RNA was extracted, and quantitative PCR (qPCR) was performed and analyzed as previously described (see Table S2 in the supplemental material) (2 (link)).

In some experiments, some of the animals were sacrificed 4 weeks after the second vaccination. Spleens were collected and splenocytes were isolated as previously described with the following modifications [13 (link)]. Splenocytes were resuspended in 96-well plates (10⁶ cells/well) in RPMI-1640 (Wisent Bioproducts) supplemented with 10% fetal bovine serum, 1 mM penicillin/streptomycin, 10 mM HEPES, 1X MEM non-essential amino acids, 1 mM sodium pyruvate, 1 mM L-glutamine (all from Wisent Bioproducts), 0.05 mM 2-mercaptoethanol (Sigma-Aldrich). The cells were incubated at 37ºC in the presence of 2.5 μg/mL of rCatB for 72 hours after which the supernatant cytokine levels of IL-2, IL-4, IL-5 IL-10, IL-12p70, IL-13, IL-17, IFNγ, and TNF-α were measured by QUANSYS multiplex ELISA (9-plex) (Quansys Biosciences, Logan, UT) following the manufacturer’s recommendations.