Guava easycyte 6 2l benchtop flow cytometer

To inhibit global Wnt secretion, mice were injected intraperitoneally with 50 mg/kg Wnt-C59 dissolved in a 5% DMSO, 30% PEG300, 1% Tween80, 64% PBS solution for 3 consecutive days prior to implantation, and every other day following surgery. To inhibit macrophageal Wnt secretion, Csf1r-iCre+; Wls^fl/fl mice were injected with tamoxifen for 5 consecutive days prior to implantation. Seven days after surgery, mice were euthanized. Cells were detached from the implant surface into single cell suspension using Accutase and washed with staining buffer for staining and flow cytometric analysis.
Prior to fluorescent staining, Fc receptors were blocked by incubation with anti-CD16/32 (BioLegend) to prevent non-specific binding of subsequent fluorescent antibodies (BioLegend). After incubation and washing, cell suspensions were incubated with fluorescent antibodies to identify macrophages (CD45+/CD68+/CD11b+), pro-inflammatory macrophages (CD45+/CD68+/CD80+), anti-inflammatory macrophages (CD45+/CD68+/CD206+), MSCs (CD11b-/Sca-1+/CD105+), CD4 T-cells (CD3+/CD4+), and CD8 T-cells (CD3+/CD8a+). Stained cell suspensions were analyzed using a Guava® easyCyte 6–2L Benchtop Flow Cytometer (MilliporeSigma, Burlington, MA) using a total of 5000 events per sample (n=6/group). Results were analyzed using FlowJo software.