Dual Infection of Macrophages with Leishmania and Borrelia

DH82 (2 × 10⁵/well) and RAW 264.7 (5 × 10⁴/well) cells were seeded in 24-well tissue culture-treated plates containing sterilized 12 mm glass coverslips (Fisherbrand, Waltham, MA, USA) and incubated at 37 °C, 5% CO₂ overnight, as described elsewhere [54 (link)]. The following day, cells were infected with L. infantum at 1:10 MOI. Two hours post-infection, wells were thoroughly washed with pre-warmed supplemented DMEM to remove non-internalized parasites and B. burgdorferi were inoculated at 1:25 MOI. 24 h and 48 h post coinfections, coverslips were fixed with methanol (Research Products International, Mount Prospect, IL, USA), stained with HEMA 3 (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Slides were assessed under a light microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan) at 1000× magnification by blinded reviewers to quantify percent L. infantum-infected cells and number of intracellular amastigotes per 100 cells.