2.5. Cell Surface Labelling Using CyDye DIGE Fluor Minimal Dye

CyDye fluorescent cell surface protein labelling was performed as previously reported [4, 20]. Briefly, approximately 20 million subconfluent PDLSC or GF were detached with either 1 mM PUCK's EDTA or 3 mg/mL type I collagenase and aliquoted into ~5 million cells per tube. Cells were washed in ice cold HBSS (pH 7.4) followed by ice cold HBSS (pH 8.5) and centrifuged at 800 ×g for 2 minutes. The cell pellets were resuspended in 200 μL labelling buffer containing HBSS (pH 8.5) and 1 M urea. Cells were then labelled with 600 pmol of either Cy2, Cy3, or Cy5 or CyDye DIGE Fluor minimal dyes on ice in the dark for 20 minutes. Staining was quenched by adding 20 μL lysine (10 mM) for 10 minutes. Surface-labelled cells were pelleted by centrifugation and resuspended in 202 μL HBSS (pH 7.4). An aliquot (2 μL) was taken prior to and after labelling to check for labelling efficiency using flow cytometry.