Quantifying Intracellular Collagen-I in Leukocytes

Flow cytometry analysis was performed as described previously in Coffey et al.33 (link). Following white blood cell isolation using density gradient centrifugation, 1×10⁶ cells were re-suspended in flow cytometry analysis buffer (Roswell Park Memorial Institute medium supplemented with 10% horse serum, 0.1% sodium azide, and 25 mmol/l HEPES). Cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) and blocked prior to intracellular staining of collagen-I with mouse antihuman collagen-I antibody (Product code MAB3391; Millipore). These were then stained with Alexa-Fluor 488 goat antimouse secondary antibody (Product code 115-545-146; Jackson ImmunoResearch Europe). Cells were finally stained for cell surface antigen CD45 using PerCP antihuman CD45 (Biolegend), re-suspended in PBS, and analyzed on the flow cytometer (BD FACSVerse)44 (link)–46 (link). All analysis was done on a BD FACSVerse (BD Biosciences) using BD FACSuite v1.0.5 (BD Biosciences). CF levels were displayed as a percentage of the total white blood cell population (Fig. 2).