CX₃CL1 rescues human blood monocytes from experimentally induced death. (A) Flow cytometric analysis of cultured human blood monocytes stained with propidium iodide (PI). Cells gated in region R7 (left dot plot) represent total analyzed population. Region R8 (right dot plot) gates PI⁺ cells. All bar diagrams shown in this figure present the portion of R8 gated PI⁺ dying cells out of R7 gated total cells. (B) Human blood monocytes (CD14⁺⁺CD16⁻) were incubated in RPMI supplemented with 10% FCS, 10 nM soluble recombinant CX₃CL1 (rCX₃CL1), or nonsupplemented medium for 4 hours; n = 3. (C) Human blood monocytes (CD14⁺⁺CD16⁻) were incubated in serum-supplemented RPMI either further supplemented or nonsupplemented with 10 nM soluble recombinant CX₃CL1 (rCX₃CL1). Six hours later, 30 μM 7-β-hydroxycholesterol (7β-OH-CH) was added to the cultures, and cells were analyzed after additional 16 hours; n = 3. (D) Human blood monocytes (CD14⁺⁺CD16⁻) were incubated with RPMI supplemented with 1, 5, 10, or 100 nM CX₃CL1. Diagram shows percentage of PI⁺ cells; n = 3. (E) Human blood monocytes (CD14⁺CD16⁺) were incubated in RPMI supplemented with 1 μg/mL pertussis toxin (PTx) for 1 hour followed by incubation with 10% FCS, 10 nM soluble recombinant CX₃CL1 (rCX₃CL1) or unsupplemented medium for additional 4 hours; n = 3. (F) Flow cytometric analysis of isolated CD14⁺⁺CD16⁻ (top dot plot) and CD14⁺CD16⁺ (bottom dot plot) human blood monocytes, before culture. Numbers indicate percentage of gated cells out of total population. (G) Isolated CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ human blood monocytes incubated in RPMI (Medium; □) or RPMI supplemented with either 10% FCS (+FCS; ■) or 10 nM recombinant CX₃CL1 (+CX₃CL1; ); n = 3. *P < .005, **P < .01, ***P < .05, NS = not significant (Student t test).