Leishman stain

PMN were collected from bone marrow of B6 and mTLR4KO mice as previously described [21 (link)]. Briefly, the femurs and tibia were removed from the mice after euthanasia and flushed with Hanks buffered Salt Solution (HBSS, ThermoFisher Scientific, Waltham, MA, USA). Cell clumps were dispersed, debris removed and centrifuged. Pellets were washed, resuspended in HBSS, and layered on top of a three-step discontinuous Percoll (Cytiva Sweden AB, Uppsala, SWE, USA) density gradient prepared in a 15 mL polystyrene tube by layering 2 mL of 75, 67, and 52% Percoll solutions. PMN fraction was collected from the lowest band (at the 75/67% interface) after centrifugation at 1647 g for 30 min at room temperature. Red blood cells were eliminated by hypotonic lysis. The purity of PMN was typically above 90%, assessed by Leishman Stain (Sigma, St. Louis, MO, USA). PMN were treated with 2 mM GLY and incubated with KEI 1025 (MOI 10) for 1.5 h as described above and mRNA levels of TLR4 and TLR9 were determined by RT-PCR.

The media and reagents used to culture BM were Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Corporation, Carlsbad, CA, USA), fetal bovine serum (FBS; JRS Scientific Inc., Woodland, CA, USA), penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Australia), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-6 (IL-6) (Miltenyi Biotec, Bergisch Gladbach, Germany). The reagent, 1,4-BQ (CAS No: 106-51-4 and ≥ 98% purity) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Methylcellulose culture media were purchased from StemCell Technologies (Vancouver, BC, Canada) for culturing each progenitor lineages. The reagents used throughout the conventional karyotyping experiments were KaryoMAX Colcemid Solution, Trypsin EDTA 1× (Gibco, Grand Island, NY, USA), and Leishman stain (Sigma-Aldrich Co., St. Louis, MO, USA).

To examine the morphology of cultured promastigotes, a Leishman stain was performed (Sigma-Aldrich) on cell-dense promastigote cultures, in accordance with the manufacturer’s instructions. Cell morphology was examined by oil emersion light microscopy (1000X magnification) using a Leica DM1000 microscope (Leica Microsystems). To examine their ultrastructural features, cultured promastigotes were embedded in low melting point agarose and prepared for transmission electron microscopy using standard procedures (S1 File). Following this, ultrathin sections were cut from the agarose and examined using a Hitachi H-7650 Transmission Electron Microscope (USA).