Fig. 1.

Quantitation of macrophages engineered to express lacZ through β-galactosidase activity. (A) Schematic representation of the MMLV retroviral vector used to generate retroviral particles to transform P388D1 and J774.1 macrophages. The human EIFα promoter provides for the constitutive expression of lacZ in transformed macrophages, which were selected by resistance to G418. IRES, internal ribosome entry site. (B) β-Galactosidase hydrolysis of ONPG is proportional to macrophage numbers. Increasing numbers of P388D1-lacZ macrophages were lysed, and β-galactosidase activity was measured as absorbance at 420 nm. Data points represent individual measurements from triplicate wells, and a line of best fit shows the linearity of the assay for the quantitation of macrophages. (C) The dose-dependent kinetics of macrophage killing by Histoplasma yeast. P388D1-lacZ macrophages were infected with wild-type G217B Histoplasma yeast at MOIs of 0.25:1 (squares), 1:1 (circles), or 4:1 (triangles) yeast to macrophages. At days 1 through 7 postinfection, remaining macrophages were assayed for residual β-galactosidase activity and normalized to that of uninfected wells (diamonds). Values are presented as averages ± standard deviations (SD) from three replicate infections. Data points were statistically compared to each other at each time point. Nonsignificant differences (ns) are indicated by boxes around the relative data points. All other points were statistically different (P < 0.05) from uninfected samples and samples infected at other MOIs.