Macsquant cytometer

Flow cytometry was employed to verify the varying levels of CD38 expression by the lung cancer cell lines. Daratumumab served as the primary antibody, at a concentration of 20 μg of antibody per 1 mL of solution, while goat anti-human AlexaFluor488 secondary antibody was utilized. Proper controls of cells alone, primary antibody alone, secondary antibody alone, and a nonspecific IgG antibody were employed. Staining and flow cytometry analysis followed standard protocols.^{24 (link)} Analysis was performed using the MACSQuant cytometer and FlowJo (V.10) software.

Keratinocytes and fibroblasts were fixed, permeabilized (BD Cytofix/Cytoperm kit), and stained with antibodies against human vimentin (BD Pharmingen clone RV202), KRT15 (Abam clone LHK15) labeled with a chromophore preconjugated to Fab (Zenon mouse IgG labeling kit) or human CD200 (BD Biosciences; 552475). Data was collected on a dual-laser flow cytometer (BD FACSCalibur) followed by FlowJo 10 (TreeStar) software analysis.
To measure TCRγδ expression, healed wounds (~WD20) from wild type and TLRKO3 mice were minced and digested at 37 C in a buffer containing RPMI 1640, 1.67 collagenase3 Wunsch units/mL Liberase TL (Roche Life Sciences, Indianapolis, IN) and .01% DNAse (Sigma-Aldrich, St Louis, MO) for 75 minutes. Following digestion, samples were washed and filtered (40μm) to obtain a single cell suspension. Cells were stained with propidium iodide (Miltenyi Biotec, San Diego, CA) and TCRγδ (GL3) antibody (Miltenyi Biotec, San Diego, CA) followed by analysis with MACSQuant cytometer and FlowJo software.