Guava easycyte 6 2l cytometer

Surface staining of single-cell suspensions of dendritic cells was performed using standard protocols (31 (link)) and analyzed on a Millipore Guava Easycyte 6 2L cytometer. Data analysis was conducted using FlowJo 10.0.7 software. The following were obtained from BioLegend: anti-CD11c-APC (147309), anti-MHC-II-PerCP-Cy5.5 (107626), and anti-CD86-PeCy5 (105014).

Twenty-four hours after treatment, the percentage of apoptotic cells was assessed using a standard flow cytometry Annexin-V-FITC binding assay (BD Pharmingen). Briefly, cells were disaggregated by trypsin digestion and washed with PBS. The pellet was incubated at room temperature with 5 μg/ml Annexin V-FITC, 5 μg/ml propidium iodide (PI) and binding buffer for 15 min in the dark. Annexin V and PI fluorescence were measured using a Millipore Guava Easycyte 6 2L cytometer. According to the manufacturer's instructions, “cells that stain positive for FITC Annexin V and negative for PI are undergoing apoptosis. Cells that stain positive for both FITC Annexin V and PI are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both FITC Annexin V and PI are alive and not undergoing measurable apoptosis.” Data was analyzed using FlowJo 10.0.7 software.