Primary cell irradiation

hTERT-MSCs were seeded at 70% confluency per well in 4 mL of RPMI-1640 medium containing 10% fetal bovine serum (FBS, Invitrogen) and 1 μM hydrocortisone (Sigma) 24 h prior to seeding with primary B-ALL or stem cells from bone marrow. To seed primary cells, RPMI-1640 complete medium was removed before adding 3.2 × 10⁶ primary cells, recovered from cryopreserved samples, in 4 mL of AIM-V medium. Both primary cells and hTERT-MSCs were incubated at 37 °C in a 5% (v/v) CO₂ incubator. After 24 h co-culture, primary cells were removed from co-culture and treated with 1 Gy X-irradiation or sham conditions. Primary cells were then added back to hTERT-MSC co-culture. Half of the primary cells were fixed 0.5hrs after irradiation, with the other half fixed 24 h after irradiation. Cells were concentrated onto a slide using the Epridia Cytospin 4 centrifuge (Fisher) and fixed in methanol at −20 °C for 5 min before storage at −20 °C.