| BALB/c spleens from naïve and tumor mice were mechanically digested and placed through a 70-μm cell strainer to obtain single cell suspensions. To avoid nonspecific staining, single cells Naïve and suture-inflamed BALB/c corneas were harvested and immersed in PBS containing 20 mM EDTA (Sigma-Aldrich) at 37°C for 30 minutes, and the corneal epithelium was removed with forceps to allow investigation of the corneal epithelium and stroma separately. Following two washes with PBS, the corneal stroma was cut into pieces and digested with 2 mg/mL collagenase D (Roche, Indianapolis, IN, USA) and 2 mg/mL DNAse I (Sigma-Aldrich, St. Louis, MO, USA) in DMEM for 60 minutes at 37°C. After digestion, corneal single cell suspensions were passed through a 70-μm cell strainer, and corneal epithelia and stroma were pooled into one and two separate samples per state, respectively, and blocked with Fc-block (as above). |
Samples were stained with fluorophore-conjugated antibodies against cell surface markers CXCR4. Single cell suspensions were then washed, fixed, and permeabilized (Cat. 555028; BD Bioscience) |
Single cell suspensions were then stained with an anti-rabbit IgG secondary antibody (Jackson ImmunoResearch), and following a final wash underwent flow cytometric analysis using BD LSR II Cytometer (BD Biosciences). Acquired data were analyzed by FlowJo v10 (FlowJo, LLC, Ashland, OR, USA). |