Fcs express 6 flow research edition

BMDMs and human macrophages were differentiated, as described above. Differentiated macrophages were resuspended in EV-free DMEM medium and aliquoted into microfuge tubes. Cells (5 × 10⁵) were incubated with 40 µg of DiO-labeled EVs, 40 µg of unlabeled EVs, a combination of 20 µg of DiO-labeled and 20 µg of unlabeled EVs, or no EVs for 45 min at 37 °C under 5% CO₂. Cells were centrifuged at 300×g for 10 min to eliminate unbound EVs. Macrophages were resuspended in 250 µl of EV-free DMEM medium and labeled with either APC-labeled anti-mouse F4/80 antibodies (Invitrogen MF48005; 1:100) or APC/Cy7-labeled anti-human CD11b (Biolegend 301341; 1:100) and APC anti-human CD14 (eBioscience 17-0149-41; 1:100) for 30 min at RT. Three replicates were left unlabeled as controls. Excess antibody was eliminated by centrifuging at 300×g for 10 min and resuspending cells in 1% paraformaldehyde in PBS. Cell fluorescent labeling was analyzed using a LSR II (BD Biosciences). Data was analyzed with FCS Express 6 Flow Research Edition (De Novo Software). Experiments were performed in the Flow and Mass Cytometry Core Facility of the University of Maryland School of Medicine Center for Innovative Biomedical Resources (CIBR).

Flow cytometry data was acquired by CytoFLEX S (Beckman Coulter) and analyzed with FCS Express 6 Flow Research Edition (DeNovo Software). The percentage of CD14 + monocytes and CD3 + lymphocytes from all CD45 + white blood cells were determined from the initial leukapheresis product and after monocyte enrichment. The cells were blocked with FcR Blocking Reagent (Miltenyi Biotec) and stained with CD45-PE, CD3-BV421 and CD14-APC antibodies and 7-AAD to assess viability. For all differentiated cells a total of 23 surface markers (CD10, CD11c, CD14, CD16, CD38, CD40, CD49c, CD51, CD71, CD80, CD83, CD85h, CD86, CD103, CD163, CD206, CD209, CD258, CD282, CD284, CD370, Syn3, VEGFR1) were determined as detailed in Supplementary Table S1. Extracellular markers were analyzed from live macrophages, defined as CD45-positive, viability dye (SYTOX Green) negative events. Normalized median fluorescence intensity (nMFI) was calculated by subtracting the nMFI for the CD45-only stain from each measurement. The normalized nMFIs were ordinated using linear discriminant analysis (LDA) using the MASS v7.3.51.4³³ package in R. For indoleamine 2,3-dioxygenase (IDO) intracellular staining cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience). The IDO signal was analyzed from CD33-positive, eFluor 660 Fixable Viability Dye-negative events.