Figure 2

Isolation and FACS of tumor cells. A. Procedure for isolation and dissociation of tumor cells into single cells. The tumor specimens were minced by using crossed scalpels to cut them into small pieces over an ice-bath. The minced pieces were triturated with 50-mL and 25-mL pipette, consecutively. The sample was washed 6X with cold Hank’s buffer-saline solution (HBSS) without phenol red and allowed to settle by gravity (3–5 min). The supernatant was transferred to a fresh 50-mL conical polypropylene tube (Falcon, Becton Dickinson) and the precipitate (necrotic tissue [black] and vessel pieces) was discarded. The pieces were washed repeatedly until the supernatant became clear. Remaining red blood cells were removed by step-gradient centrifugation over Histopaque-1077. The pellet was red blood cells and the brain tissue was in the supernatant. The supernatant was washed with HBSS and centrifuged (183 g, 5 min, 3x) to remove the Histopaque-1077. The pellet was triturated sequentially with 10 mL, 5 mL, and 2 mL pipettes. The suspension was then digested with collagenases, papain, protease, DNase, and Dispase II. The loose cells were washed and the cell pellet was suspended in cell dissociation buffer. B. FACS analysis of tumor cells. The surface marker expression (CD133, CD29, CD34) were used. The antibodies were as for name/synonym/clone: CD29/integrin-β1/MAR4, CD34/Sialomucin-I/AC136, and CD133-1/Prominin-1/AC133.