Fc receptor blocking solution

Human CD14⁺ monocytes were evaluated for expression of VEGFR-3, LYVE-1 and PDPN at time of isolation (ex vivo), after 10 days of treatment with CSF1, and on day 14 after being grown with TLR4 ligands for 4 days. Monocytes were first incubated with Fc receptor blocking solution (BD Biosciences, San Jose, CA) followed by incubation with 2 μg/ml of anti-VEGFR-3, -LYVE-1 and -PDPN antibodies for 30 minutes on ice before they were washed with ice-cold F-Buffer (DPBS containing 2.5% horse serum and 0.02% sodium azide) and incubated with secondary antibodies conjugated to Dylight 488- or 650. Cells from at least three healthy donors were used for each experiment with replicates performed in duplicate or triplicate. Data were acquired with an AccuriC6 flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, OR). Results are expressed as mean percentage (%) of cells positive for marker expression ± standard error mean (SEM) or when applicable, mean fluorescent intensity (MFI) ± SEM. Cells treated with CSF1 alone in the absence of TLR4 ligands served as controls.

After isolation, cell pellets were resuspended in FC receptor blocking solution (553141, BD Biosciences), incubated on ice for 10 min, and costained for 30 min on ice in the dark with PE-Cy7-labeled CD45 (103114, BioLegend), PE-labeled CD11b (101208, BioLegend), and APC-labeled ACSA2 (130-117-386, Miltenyi Biotec). The cells were then rinsed in PBS, centrifuged, resuspended in FACS buffer (1% FBS + 2 mM EDTA, 25 mM HEPES, 1:500 RNA inhibitor in PBS), and incubated in 7AAD (420403, BioLegend) for 10 min before sorting. Data were analyzed using the BD FACS Diva v8.0.1 software. Sorted cells were centrifuged at 400×g for 10 min and pellets were lysed in RLT-buffer (74004, Qiagen) for RNA extraction.