<i>B. burgdorferi</i> incorporate BODIPY-cholesterol into its outer membrane as a component of the cholesterol-glycolipids.

<p><b>A.</b> Spirochetes were grown for 6 hrs in BSK-II media lacking cholesterol and their fluorescence was measured by spectrophotometry. 4.0 mg/L BODIPY-cholesterol in BSK-II without free cholesterol (green); 0.25% DMSO in BSK-II without free cholesterol (purple); 2.0 mg/L BODIPY-cholesterol BSK-II without free cholesterol (red); 0.175% DMSO in BSK-II without free cholesterol (dark blue); 0.2 mg/L BODIPY-cholesterol in BSK-II without free cholesterol (light blue); 0.0125% DMSO in BSK-II without free cholesterol (black). <b>B... </b><i>B. burgdorferi</i> incubated with and without BODIPY-cholesterol were analyzed by flow cytometry. ANOVA ***p<0.001. <b>C.</b> Unfixed, live spirochetes grown in the presence of 4.0 mg/L BODIPY-cholesterol for 4 hrs were observed by fluorescent microscopy. The spirochetes did not exhibit morphological or motility defects. Scale bar = 20 µm. <b>D.</b> Following incubation of <i>B. burgdorferi</i> with 4.0 mg/L BODIPY-cholesterol for 4 hrs, the spirochetes were grown in BSK-II for up to 96 hrs, and there were no significant differences in growth compared to controls. Untreated control (black diamond); labeled with BODIPY-cholesterol (blue square); Incubated with 0.25% DMSO (red triangle). <b>E... </b><i>B. burgdorferi</i> were treated and incubated with 4.0 mg/L. <i>B. burgdorferi</i> pellets were extracted by Bligh and Dyer lipid extraction and analyzed using a chloroform-methanol (85/15) HPTLC. The HPTLC plate was developed using iodine and exposure to UV light. <b>F... </b><i>B. burgdorferi</i> were grown in BSK-II media lacking free cholesterol with BODIPY-cholesterol or cholesterol as the primary source of sterol in the media to assess long term growth and cytotoxicity in the presence of exogenously added sterol. BSK-II (green); 4.0 mg/L BODIPY-cholesterol in cholesterol free BSK-II without serum (blue); 4.0 mg/L cholesterol in cholesterol free BSK-II without serum (black). Experiments <b>A, B, D,</b> and <b>F</b> represent the mean ± standard error of the mean from three separate experiments.