Schneider s drosophila medium

Promastigote stage of L. infantum (MHOM/TN/80/IPT1), L. tropica (MHOM/SY/2012/ISS3130) and L. braziliensis (MHOM/PE/2006/ISS2848) were cultured in Schneider’s Drosophila Medium (Lonza) supplemented with 10% heat-inactivated fetal calf serum (HyClone), 20 mM Hepes, and 2 mM L-glutamine at 23 °C.

Prior to testing, ticks were washed with 70% ethanol and then rinsed with Schneider's Drosophila Medium (Lonza, Walkersville, MD, USA) containing 200 IU/ml penicillin, 200 μg/ml streptomycin and 0.5 μg/ml amphotericin B. Ticks were then cut into small pieces and snap-frozen in liquid nitrogen for 5 min before the samples were mixed with 500 μl of Dulbecco's Modified Eagle's medium (DMEM) The samples were then lysed using a Qiagen Tissue Lyser with 3 mm Tungsten beads at 25 Hz for 2 × 30 s. A further 500 μl of DMEM was added to the samples, followed by centrifugation at 600 × g for 5 min at room temperature and then supernatant was collected. One half of the supernatant was used for PCR; DNA was extracted using a previously-described DNA extraction method (Tuppurainen et al., 2011 ) followed by conventional PCR (Tuppurainen et al., 2011 ) or the real-time PCR (Bowden et al., 2008; Stubbs et al., 2012 ) as described below for testing the cell culture samples. In the real-time PCR, cycle threshold (C_t) values over 39 were considered negative. The other half of the supernatant of each sample was used for virus isolation (Lubinga et al., 2014c (link)). Tick samples collected from Egypt in 2006 did not contain sufficient material for virus isolation.

African green monkey kidney Vero E6 cells (ATCC CRl-1586) were cultured at 37°C with 5% CO₂ in Dulbecco’s Modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/ml; Sigma-Aldrich) and streptomycin (100 μg/ml; Sigma-Aldrich). Ae. albopictus C6/36 (ATCC CRL-1660) cells were cultured at 28°C in Leibovitz L-15 medium (Gibco) supplemented with 10% FBS, 2% tryptose phosphate broth (Gibco) and 1% nonessential amino acids (Gibco). Ae. aegypti Aag2 cells were cultured at 28°C in Schneider’s Drosophila medium (Lonza) supplemented with 10% FBS. During experiments gentamicin (50 μg/ml; Life technologies) was added to the culture medium of C6/36 and Aag2 cells.