Diff quick stain

After donation, blood was immediately processed as previously described [21 (link)]. Briefly, RBCs were separated from plasma and leukocytes by three washings in wash medium [RPMI 1640 (Invitrogen) containing 2 mM glutamine, 25 mM HEPES, 20 mM glucose, 27 μg/mL hypoxanthine and 32 μg/mL of gentamicin (Sigma) (pH 7.2)]; P. falciparum strain Palo Alto was then cultured at 1–5% hematocrit [33 (link)] under a 1% O₂, 5% CO₂, and 94% N₂ atmosphere in complete media (CM) [wash medium supplemented with 0.5% Albumax II (Gibco)]. Parasites were synchronized using a Percoll gradient method, as previously described [24 (link)]. After allowing the synchronized parasites to mature and reinvade fresh RBCs, drug studies were performed at the desired times as hours post-invasion (hpi). To assess parasitemia and infected cell morphology, thin smears were prepared, labeled with Diff-Quick stain (Siemens), and examined by light microscopy.

Nasal airway lavage (NAL) on mice and analysis of infiltrating inflammatory cells were performed using the trans-pharyngeal nasal lavaging technique developed by our laboratory [19] (link). Different from previously used trans-tracheal technique [27] (link), this technique minimizes cells loss and gives consistent cytology results [19] (link). Briefly, the choana was cannulated with 24G catheter through the pharyngeal opening above the vocal cord by transpharyngeal approach. The nasal cavities were gently lavaged with 350 µL of cold PBS twice and the fluid from the nostrils was collected. NAL fluids were centrifuged, and supernatants were stored at −80°C until assayed. Cell pellet was resuspended with 100 µL PBS and the total cells were counted using hemocytometer. Cytospin slides were prepared and stained with Diff-Quick stain (Dade Behring, Deerfield, Illinois), followed by differential cell count of at least 200 cells per slide.

Ten milliliters of human peripheral blood from a healthy volunteer was centrifuged at 4°C, 1400 × g for 10 minutes to purify the buffy coat (leukocyte layer). The buffy coat was overlaid on OptiPrep™ (Alere Technologies AS, Oslo, Norway) density-gradient media that had been adjusted to a specific gravity of 1.077 g/cm³. This was centrifuged at 20°C, 800 × g for 20 minutes to separate the mononuclear cells. After washing twice with phosphate-buffered saline (PBS), the mononuclear cells were incubated with anti-human CD14-FITC-labeled antibody and anti-human CD19-PE-labeled antibody (Thermo Fisher Scientific, Inc., Waltham, MA) at 4°C for 20 minutes.
After washing, CD14⁺/CD19⁻ cells were sorted by flow cytometry (FCM) using FACSVantage SE (BD Bioscience, San Jose, CA), and collected to recover purified monocytes (Kanai et al., 2007 (link)). Cell morphology of the sorted CD14⁺/CD19⁻ cells was confirmed using Diff-Quick stain™ (Dade Behring, Inc., Deerfield, IL). All procedures were performed in compliance with the Recombinant DNA Experiment Safety Committee, Fujita Health University (DP16051).