Brain tumor tissue dissociation

The tissue specimen was weighed, cut into ∼1 mm³ pieces with a sterile razor blade, re-suspended at 1,600 mg tissue/10 mL in Hibernate-A medium ⁵⁵ (Thermo Fisher) containing 1% Pen/Strep and primocin (10 µg/mL, InvivoGen, San Diego, CA, USA). The tissue suspension was treated with a cocktail of enzymes (DNase I, 50 U; dispase II, 5 U; collagenase I, 1 U and collagenase IV, 10 mg/mL in 10 mL 0.5% Trypsin-EDTA solution) at 37°C for 20 min, followed by neutralization with a 10 mL trypsin inhibitor (0.5%, w/v) solution and gentle pipetting. The tissue dissociation solution was filtered with 100-µm cell strainer (Fisher Scientific, Suwannee, GA, USA) and single cell suspension was collected.