Isolation of CD14+ Cells from Human Blood

Fresh human peripheral blood (n = 3) from healthy donors and from active RA patients (deidentified) was collected after institutional review board (IRB) approval and written consent from donors from The Ohio State University Medical Center, and processed following an earlier-described protocol [18 (link),31 (link)]. In, brief, peripheral blood mononuclear cells were isolated from 40 mL of freshly collected blood using Ficoll-Paque density centrifugation. After removing platelets from the peripheral blood mononuclear cell (PBMC) by washing with PBS, CD14⁺ cells were isolated using an AutoMACS device, and magnetic bead conjugated CD14⁺ antibody and reagents (all from Miltenyi Biotec, San Diego, CA, USA) following an earlier-established protocol [18 (link),19 (link)]. Isolated CD14⁺ cells were used for total RNA was extraction and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis.