Grace s medium

C57BL/6J (2–6 months old) mice were obtained from the Universidade Federal de Ouro Preto animal facility. Animals received water and food ad libitum. This study was carried out in accordance with the recommendations of the Brazilian Guidelines for animal experimentation. The protocols were approved by the University’s Ethical Committee on Animal Experimentation (CEUA 2012/02 and CEUA 2013/51). Leishmania amazonensis, PH8 strain (IFLA/BR/67/PH8) promastigotes were grown in Grace’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS, Cultilab, Campinas, SP, Brazil), 2 mM l-glutamine (Sigma-Aldrich) and 100 U/mL penicillin G potassium (Sigma-Aldrich), pH 6.5, at 25°C. Metacyclic promastigotes were purified by gradient centrifugation of parasites at the stationary phase of culture (day 5) over Ficoll 400 (Sigma-Aldrich), as previously described (5 (link)). In in vitro DC infection experiments, metacyclic promastigotes, suspended in PBS with 5% FCS, were incubated in the presence of 5 µM CFSE (Sigma-Aldrich) at 37°C for 10 min in the dark. The suspension was centrifuged and the parasites were washed in PBS, pH 7.2 (37 (link)). Alternatively, parasites were labeled with PKH26 (Sigma-Aldrich) according to the manufacturer’s instructions.

To infect ants with O. unilateralis s.l.,
fungal colonies were placed in a sterile 2 mL tube with two 8/32 inch metal balls
(Wheels Manufacturing Inc.) and 500 μl Grace’s medium (Sigma) freshly supplemented
with 10% FBS (PAA laboratories Inc.). The colony was disrupted using a TissueLyser
II (Qiagen) at RT for 60 sec. at 30 freq/sec. This way single hyphae were obtained
that were used at a mean concentration of 3.9×10⁷ +/−
1.1×10⁷ hyphae/ml. Infections were done by injecting
1 μL hyphal solution with a laser pulled 10 μL micropipette (Drummond) and
aspirator tube (Drummond) into the thorax underneath the front legs. Sham
treatments were done in similar fashion using 1 μL medium without hyphae. To test
the effect of the two candidate compounds identified in this work, commercially
obtained standards were injected alone or in combination using the same method.
Serial 10 times dilutions between 1 μg/ml and 100 pg/ml were made in Grace’s
medium with FBS. For the combination both equal concentrations and a series of
dilutions in opposite directions were combined (100 μg/ml sphingosine with
100 pg/ml GBA, 10 μg/ml sphingosine with 1 ng/ml GBA, etc.). Since sphingosine was
dissolved in ethanol, shams were injected with ethanol concentrations between 10%
and 0.00001%.

BmN cells were maintained in TC-100 medium (BIOTECH, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco). Sf9 cell line was cultured in HyClone SFX medium (Thermo Scientific) supplemented with 5% FBS. Cells were pre-incubated for 1 day before further experiments. For ex vivo experiments, newly collected prothoracic gland and fat body tissues from L5D2 larvae were cultured in Grace’s medium (Sigma-Aldrich, 11300-043) at 25 °C. After pre-incubation for 1 h, the medium was replaced with fresh medium. To determine which BmE75 genes are 20E-primary response genes, 2 μM 20E or/and 10 μg/ml CHX (Enzo Life Science, ALX-380-269) were added. After incubation for the indicated times, mRNA and protein were isolated for qPCR analysis and Western blotting.