Facs aria system

All the CD44⁺/CD105⁺ subpopulation of cells were collected and washed with PBS by centrifugation. The cells were suspended at a density of 1 × 10⁴ cells/ml. The cell suspension was incubated with a primary antibody recognizing a cell surface antigen or isotype control antibody (CD29, CD90, or CD105 conjugated with FITC; eBioscience) on ice in Dulbecco’s PBS (DPBS) containing 10% bovine serum albumin (BSA). The sensitivity of the flow cytometer was adjusted using the cells treated with an isotype control antibody (mouse IgG1-FITC, eBioscience) to rectify non-specific binding. Antibody staining by FCM was analyzed on a FACS Aria system (Quanta SC, Beckman Coulter INC., Indianapolis, IN, USA).

Mouse peripheral blood mononuclear cells from each group were suspended (1 × 10⁶ cells/ml) and stained with primary antibodies on ice in Dulbecco’s phosphate-buffered saline containing 10% bovine serum albumin. Staining with an isotype control antibody (mouse IgG1-FITC, mouse IgG1-PE; eBioscience™; Invitrogen, Waltham, MA, United States) was performed to correct for non-specific binding. Antibody staining was analyzed by fluorescence correlation microscopy using the FACS Aria system (Quanta SC; Beckman Coulter, Brea, CA, United States).