RCC Sphere Immunophenotyping Protocol

RCC cells were counted and seeded at density of 100 cells/well in ultra-low attachment 24 wells plates (TC plate, suspension, F, Sarstetd, Numbrecht, Germany) supplemented with sphere promoting media as described previously^{20 (link),77 (link)}. Later culture media was removed, and tumor spheres were rinsed briefly in PBS (3 times). 10% goat serum in PBS was used for blocking at room temperature (1 hr). Tumor spheres were incubated separately with diluted primary antibodies against CD105 (1:1000), CD133 (1:500), CD44 (1:1000) and CXCR4 (1:1000) at 4 °C for 4 h; washed three times with PBS and incubated with Alexa Fluor 488 secondary goat anti-mouse antibody (1:400) for 1 h at room temperature. The spheres were rinsed 3 times with PBS, followed by incubation with DAPI (1:5000; ThermoFisher Scientific, Massachusetts, USA) for 10 min. As control the spheres were incubated only with secondary antibody. The slides were washed with PBS and covered with coverslips using CoverGrip Sealant (Biotium, California, USA), and images were captured using an Olympus CKX41 fluorescence microscope.