Evaluating Mincle Signaling in Pneumocystis Interactions

To determine Mincle based cellular signaling following interaction with Pneumocystis, T-cell hybridoma line B3Z containing the lacZ β-galactosidase reporter and control constructs were employed as previously described (39 (link)). Briefly, 1 X 10⁵ B3Z/FcRγ or B3Z/FcRγ expressing Mincle cells per well were cultured in a 96-well culture plate and incubated for 18 h with Pc organisms at the concentrations indicated. This cell based reporter construct mediates generation of lacZ activity following ligation of Mincle with relevant ligands and subsequent FcRγ activation. The lacZ activity was measured in total cell lysates using chlorophenol red-β-D-galactopyranoside (Roche) as a substrate with the OD 560–620 being measured using a microtiter plate reader (Molecular Devices).