Diff quick stain

The total cell count of BAL was determined using a Malassez hemocytometer (Hycor Biomedical, Indianapolis, IN, USA). Differential cell counts were done on cytocentrifuge preparations (Cytospin 3; Shandon Scientific, Cheshire, UK) stained with Diff-Quick stain (Baxter Diagnostics, McGaw Park, IL, USA).

Bronchoalveolar lavage fluid was harvested by cannulation into the trachea and airways and centrifuged (400 × g, 5 min, 4°C). The collected BALF cells were washed and suspended in PBS and then used by fluorescence-activated cell sorting (FACS). For cytological analysis, cells from the BALF were collected on glass slides by centrifugation (400 × g, 4 min), fixed, stained with Diff-Quick^® Stain (Baxter Healthcare Corp., Miami, FL, USA), and the number of neutrophils was counted.

C57BL/6 murine bone marrow derived macrophages were cultured in RPMI medium containing 10% FBS macrophage colony-stimulating factor (20 ng/ml, ProSpec, Israel), plated in 0.5 ml on eight-chamber Lab-Tek tissue-culture slides (Miles Laboratories). The differentiated macrophages were infected with LdWT, LdCen^−/−, LdMIF^−/− and LdCen^−/−MIF^−/− stationary phase promastigote cultures (10:1 parasite-to-macrophage ratio). Free extra cellular parasites were aspirated after 6 h incubation at 37 °C in 5% CO2 and the cultures were incubated in macrophage culture medium for 7 days. The parasite counts were measured by using Diff-Quick Stain (Baxter Healthcare Corporation). A minimum of 300 macrophages were counted. The results are expressed as the number of amastigotes per 100 macrophages. The experiment was performed four times (n = 3/replication), and cells were pooled.