Gibco pb max karyotyping medium

Peripheral blood was drawn and shipped in 10 mL sodium heparin tubes (Becton, Dickinson and Co, Franklin Lakes, NJ, USA; #367874) under ambient conditions to Colorado State University and received within 24 h of blood draw. All heparinized blood samples were cultured in T-25 tissue culture flasks, at 1 parts blood per 9 parts Gibco PB-Max Karyotyping Medium (Thermo Fisher, Waltham, MA, USA; #12557013), with 5.0 mM 5-bromo-deoxyuridine (BrdU) and 1.0 mM 5-bromo-deoxycytidine (BrdC) added to the medium as previously described [62 (link)]. Pre-IMRT blood samples were split into two fractions (non-irradiated and in vitro irradiated) with identical culturing conditions as other time point samples; irradiated fraction was exposed in vitro at a dose rate of 2.5 Gy/min for a total dose of 4 Gy (¹³⁷Cs gamma-ray Mark I irradiator; J.L. Shepherd & Associates, San Fernando, CA USA). Forty-eight hours after stimulation, KaryoMax Colcemid (Thermo Fisher, Waltham, MA, USA; #15210040) was added (0.1 μg per mL of medium) for four hours of incubation, then metaphase chromosome spreads were harvested with standard cytogenetic protocols [71 (link)]. Prior to Telo-FISH and dGH, slides with metaphase chromosome spreads were subject to CO-FISH protocol for removal of BrdU/BrdC incorporated DNA as previously described [72 (link)].