Figure 3.

Repletion with raft-altering sterols affects phagocytosis.A, macrophages (J774.1) were pretreated with 10 mM methyl-beta-cyclodextrin (MβCD) to deplete cholesterol and then washed and incubated with 2.5 mM MβCD loaded with 0.2 mM of indicated sterol. Monolayers were then washed and subject to lipid extraction. Lipid extracts were then analyzed using GC-coupled MS and normalized to protein found in the cell extract by the Bradford assay (n = 3). B, macrophages were pretreated with 10 mM MβCD to deplete cholesterol and then washed and incubated with 2.5 mM MβCD loaded with 0.2 mM of indicated sterol. Monolayers were then washed and allowed to interact with antibody-opsonized Cryptococcus neoformans H99 at a 1:1 ratio for 2 h. Cells were then fixed and stained with Giemsa, and phagocytic index was calculated by microscopic observation (n = 3). Error bars represent SEM, and statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Not significant (ns), ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001 compared with the cellular cholesterol for untreated control. ^★p < 0.05 compared with the cellular 7-dehydrocholesterol for untreated control. ^◆◆P < 0.01 compared with the cellular coprostanol for untreated control. All p values were adjusted for multiplicity.