Figure 1.

Monocyte-derived macrophage (MDM) segregate into two distinct subsets: Non-adherent Siglec-1^hiCD4⁺MDM and adherent Siglec-1^LoCD4⁻MDM. Triplicate wells of primary human monocytes from nine human immunodeficiency virus (HIV)-seronegative donors (#048, #130, #170, #008, #002, #202, #132, #124, and #040) were differentiated into MDM following in vitro culture in M-CSF media for 5 days. Cultures were harvested, stained, and analyzed by flow cytometry. (A) Plots show percentage of Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM within the gated CD14⁺ cells. A representative plot of one of the two independent experiments is shown. (B) Plots show that gated CD14⁺ cells are not contaminated with CD3⁺ T cells. (C,D) In vitro cultures of M-CSF-derived MDM contain adherent and non-adherent MDM. (C) Adherent and non-adherent MDM were pooled, stained, and the gated CD14⁺ cells were sorted into Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM. (D) Non-adherent MDM were separated from adherent MDM following repeated washing with media. Adherent MDM were detached with Accutase. Both fractions were washed, stained, and the gated CD14⁺ cells were analyzed separately for the expression of Siglec-1 and CD4. Plots show that non-adherent fraction represented the Siglec-1^hiCD4⁺MDM subset, whereas the adherent fraction comprised the Siglec-1^LoCD4⁻MDM subset. (E) Histograms show the expression of CCR5 and CD163 on the gated Siglec-1^LoCD4⁻MDM (blue) and Siglec-1^hiCD4⁺MDM (red) subsets. Values in the histograms denote the mean fluorescent intensity (MFI) of the specific receptors for each of the subsets. Each experiment was done twice in triplicate and the data from one of the two experiments are shown.