Diff quik

VEGF-induced EPC migration was measured using a modified Boyden chamber as previously described [29 (link)]. In brief, 100 ng/mL of vascular endothelial growth factor-A (VEGF) was placed in each well of a Boyden companion plate. An 8 μm (pore size) insert was placed in each well containing 500 μL of EPC suspension (5 × 10⁵ cells/mL = 250,000 cells/insert). After 4 h, each Boyden chamber insert was washed, and cells were fixed and stained using DiffQuik (Sigma). The membrane was removed and mounted on a slide for quantification using light microscopy with a 20X objective.

RH, GT1, Prugniaud (Pru), VEG strain T. gondii; and Nc1 [75] (link), Nc2 [76] (link) and NcLiv strain N. caninum were maintained by serial passage in human foreskin fibroblast (HFF) monolayers as described previously [77] (link). RH stably expressing mCherry were obtained from Dr. Anita Koshy [78] (link). Primary bovine fibroblasts were provided by Dr. Kenneth John McLaughlin (Penn). Cryopreserved bone marrow cells recovered from Myd88^−/−, Myd88/Trif^−/−, and Tlr2/Tlr4^−/− mice were differentiated into macrophages as described previously [79] (link), and infected with a 1∶1 MOI of parasites for QPCR and microarray experiments at 16–22 hours post-infection. Ifnar1^−/− and Tlr3^−/− mice were provided by Drs. Hao Shen and Yongwon Choi (Penn), respectively. For mouse infections, WT and Ifnar1^−/− mice received either control IgG or anti-IFN-γ (clone XMG-1.2, 1mg I.P., BioXCell) on day 0 and again at 4 days post-infection. Mice were infected with 3×10⁶ NcLiv strain Neospora caninum by I.P. injection on day 0. All mice were maintained at the University of Pennsylvania in accordance with Institutional Animal Care and Use Committee guidelines. For cytospins, 200,000 cells were spun onto glass slides using a Shandon Cytospin and stained with Diff-Quik, dehydrated, mounted with Permount (Sigma Aldrich), and images captured on a Nikon E600 upright microscope.

Human neutrophils were isolated by dextran sedimentation followed by plasma-Percoll gradient centrifugation from whole blood of healthy volunteers as previously described (25 (link), 26 (link)). Written informed consent and ethical approval from the South Sheffield Research Ethics Committee (study number STH13927) were obtained. The purity of isolated neutrophils was determined from Diff-Quik (Sigma-Aldrich, St. Louis, MO) stained cytocentrifuge preparations by light microscopy. Neutrophils were suspended at 5 × 10⁶/ml in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, United States) + 10% fetal calf serum (FCS, PromoCell, Heidelberg, Germany) and cultured in 96-well plates at 37°C, 5% CO₂. Phagocytosis assays were performed by incubating neutrophils (in RPMI + 10% FCS) with S. aureus strains at a multiplicity of infection (MOI) of 10 for 1 h after which they were cytocentrifuged (Cytospin, Shandon) onto microscope slides (27 (link)). Cells were stained with Quik-Diff dyes and S. aureus visualized within neutrophils by oil immersion light microscopy. The phagocytic index was calculated using the following formula: (total number of engulfed bacteria/total number of neutrophils) × (number of neutrophils containing bacteria/total number of neutrophils) × 100.