Lucifer yellow ch dipotassium salt

The internalization of Nef into MΦ was quantified by flow cytometry on an LSR II (BD Biosciences) using the FlowJo software (Tree Star, Ashland, OR, USA). MΦ were incubated with FITC-labeled Nef (purchased from Fitzgerald, Acton, MA, USA) at 37 °C, detached from the wells using 0.25% trypsin (Wako) after extensive washing with PBS, and immediately subjected to flow cytometric analysis.^{14 (link)} The macropinocytosis activity of MΦ was analyzed by measuring the uptake of lucifer yellow.^{36 (link)} MΦ were incubated with lucifer yellow CH dipotassium salt (Sigma; 100 μg/ml) for 2, 4, or 6 h, detached from the wells using 0.25% trypsin after extensive washing with PBS, and immediately subjected to flow cytometric analysis. The surface expression of CD163 on MΦ and M-CSF receptor on RAW264.7 cells was also assessed by flow cytometry. Cells left untreated or stimulated with Nef were detached from the wells using enzyme-free cell dissociation buffer (Gibco, Grand Island, NY, USA), stained with PE-labeled anti-human CD163 (GHI/61; BioLegend, San Diego, CA, USA), or PE-labeled anti-mouse M-CSF receptor (AFS98; eBioscience, San Diego, CA, USA) and analyzed on the LSR II using the FlowJo software.