Schneider insect medium

Neutrophils were infected in vitro with L. infantum promastigotes stationary-phase at a ratio of 1:2 (neutrophil:parasites). For assays of cell death, mouse and human neutrophils were pretreated for 30 min with zVAD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) or zIETD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) to block caspase activation before infection. In some experiments, Nec-1 (50 µM), GSK’872 (3 µM), or NSA (10 µM), necroptosis inhibitors (all from Merck Millipore’s Calbiochem^®, Darmstadt, Germany) were used. DMSO (vehicle) 0.4% (Cayman Chemical; Ann Arbor, MI, USA) was used as control. After 18 h, mouse infected neutrophils, or after 3 h, human infected neutrophils, were centrifuged, supernatants containing noninternalized promastigotes were collected, and medium was replaced by 250 µl Schneider insect medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 20% inactive FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. After that, infected neutrophils were cultured at 25°C for an additional 3 days and intracellular load of L. infantum was estimated by production of proliferating extracellular motile promastigotes in Schneider medium (43 (link)).

The S2 cells were cultured at 28 °C with Schneider Insect Medium (Sigma, Shanghai, China, 200031) supplemented with 10% FBS in 96-well plates for 24 h. We then transfected 50 ng of firefly luciferase reporter gene plasmid, 15 ng of pRL-TK renilla luciferase plasmid, and 50 ng of expression plasmid (the pAC5.1-Basic plasmid was used as a negative control) into each well of S2 cells using Lipofectamine 3000 Transfection Reagent (ThermoFisher, Waltham, MA, USA) following the manufacturer’s instructions. Fluorescence intensity was measured at 48 h post-transfection. LPS was added to the cells at a concentration of 5 μg/mL for a period of 6 h [22 (link),23 (link)]. All experiments were repeated three times.

D. melanogaster S2 cells [41] (link) were grown in Schneider insect medium (Sigma) with 10% (v/v) fetal bovine serum. DNA transfections were performed using Effectene (Qiagen). Cells were harvested 30 h post transfection and total RNA was isolated using Trizol (Invitrogen).