Countess 2 fl automated

All HEK 293T cells were enumerated and viability was determined using a hemocytometer and 0.4% trypan blue vital dye solution (Thermo Fisher Scientific, Waltham, MA, USA) as well as an Invitrogen Countess™ II FL automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA) [45 (link)]. Cell viability assessment using the trypan blue assay began within 1 h after freeze/thaw and was completed within 12 h.

BT474 breast cancer cells were purchased from ATCC (Manassas, Virginia). Cells were cultured in improved minimal essential medium (IMEM, Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 μg/mL insulin. Cells were grown at 37 °C with 5% CO₂. Cells were cultured to 70–80% confluency and all cell counts were determined by the Countess II FL automated cell counter (Thermo Fisher Scientific Inc., Waltham, MA).

mAb producing cell pools and clones were cultured in PowerCHO-2CD medium supplemented with GSEM and various concentrations of MSX (0, 25 and 50 μM). Exponentially growing cells were inoculated at a concentration of 3 × 10⁵ cells/mL into 125-mL Erlenmeyer flask containing 50 mL of culture medium. The flasks were incubated in a Climo-shaking incubator (Kuhner Ag, Basel, Switzerland) at 110 rpm in a humidified 5% CO₂/air mixture at 37 °C. Viable cells were distinguished from dead cells using the trypan blue dye exclusion method, and the viable cell concentration (VCC) was estimated using a Countess II^® FL automated cell counter (Thermo Fisher Scientific).
The long-term culture was done by continuous sub-culturing of the mAb producing clones for 30 passages (a period of approximately three months). Exponentially growing cells were inoculated at a concentration of 3 × 10⁵ cells/mL into 125-mL Erlenmeyer flask containing 30 mL culture medium and were sub-cultured every three days. At every passage, the VCC was evaluated, and culture supernatants were collected and kept frozen at −70 °C for further analyses. At passage 0, 10, 20 and 30, genomic DNA and total RNA were prepared for the analysis of the relative gene copy number and mRNA level.

The rate of NHEJ repair was quantified using U87-MG and T98G cells expressing the EJ5-GFP reporter, as described previously [29 (link)]. Briefly, cells stably expressing the reporter gene were obtained by transfection with Lipofectamine 3000 followed by selection and clone isolation with 5 μg/mL puromycin. For the NHEJ assay, the clones were transfected with the I-SceI expression vector or empty vector, and 72 h after transfection, the percentage of GFP-positive cells was determined by the Thermo Fisher Countess II FL automated cell counter.