Automated imaging system

To analyze SCAs-formation, mFISH analysis and classical cytogenetic analysis were employed. Briefly, 2.5 × 10⁶ cells transfected with pCMV-3xNLS-ISceI plasmid were split in three dishes and plated for 24, 30 and 48 h, respectively. To accumulate cells at metaphase, colcemid (Biochrom AG) was added for 2–3 h at a concentration of 0.1 μg/ml. Metaphase spreads were prepared using standard procedures. mFISH was performed using 12XCHamster Multicolor FISH Probe for Chinese Hamster Chromosomes (MetaSystems Probes) according to manufacturer’s protocol. An automated imaging system (MetaSystems) was used to obtain high quality images of metaphase chromosomes, as previously described (Soni et al., 2019 (link)). For analysis, at least 100 metaphases were scored in each of three independent experiments. The number of the SCAs formed in the non-transfected clones is subtracted from the SCAs number in cells transfected with I-SceI expressing plasmid.
Classical cytogenetic methods were also employed, as previously described (Schipler et al., 2016 (link)). High quality images of metaphase chromosomes were captured using Zeiss AxioScan.Z1 imaging platform at a magnification of ×40 dry objective. Images were analyzed using the integrated ZEN software. For analysis, at least 100 metaphases were scored in each of three independent experiments.

Lineage depleted cord blood cells were thawed and CD34⁺ CD38⁻ cells were sorted using CD34 APC-Cy7 (clone 581, custom conjugation) and CD38 PE-Cy7 (335825, HB7). Cells were pre-cultured for 36–48 h in serum-free media and subsequently electroporated as described above. After overnight incubation, media was changed to IMDM (Thermo Fisher, 12440061) with 10% FBS (Sigma, 15A085), 1× l-Glutamine (Thermo Fisher, 25030081), 1× Penicillin–Streptomycin (Thermo Fisher, 15140122) and the following cytokines (all from Miltenyi Biotec): FLT3L (100 ng/mL), G-CSF (10 ng/mL), SCF (100 ng/mL), TPO (15 ng/mL), and IL-6 (10 ng/mL) to allow for cell expansion. Subsequently, karyotyping of chromosomes was performed according to standard procedures. Metaphase slides were prepared, then banded with trypsin and stained with Leishman’s stain. The G-banded slides were scanned and metaphases captured using an automated imaging system (MetaSystems). Metaphases were analyzed using Ikaros image analysis software (MetaSystems). For each condition, 20 metaphases were analyzed by G-banded karyotyping for numerical and structural abnormalities.