Fcs express

For the establishment of a three dimensional model, the bench-top bioreactor BioLevitator (Hamilton Company, Switzerland) and basal membrane coated GEM were used. Cells were incubated in specialized culture vessels (LeviTubes; Hamilton)) in the bioreactor at 37°C and 5% CO2. 3×10⁶ cells were seeded on 1 ml GEM pre-coated with collagen IV or gelatine (Omni Life Science, Bremen, Germany) in a total volume of 10 ml medium with 10% FBS. After an overnight inoculation period, LeviTubes were filled with an additional 15 ml of medium. An aliquot of the microcarrier cultures was treated with ultrasonic irradiation, 1 h later GEM were dissolved in pre-warmed trypsin/EDTA, GEM were removed by a magnet, the restraining cells were stained with PI measurement and analysed by FACSCalibur (BD Biosciences, San Jose, US) and by FCSexpress De Novo (Glendale, CA) as described above. Staining was additionally documented on a LSM510 Meta confocal laser scanning microscope (Zeiss, Germany).

To assess the toxicity of the small molecules, mESC cells with stably integrated gRNAs from the 48-site library were transfected with p2T CAG Cas9 BlastR and KU-0060648, KU-60019, or DMSO as indicated. After 24 h of transfection, cells were trypsinized, stained with 0.5 µg/mL propidium iodide (PI, Biolegend), and subjected to flow cytometry using a Cytek DXP11 using a consistent flow-rate. Data were analyzed using BD FACSDiva and FCS Express software, and the PI gating parameters were obtained using a live-cell gate set using untreated cells and 0.25% Triton X-100-treated cells (Supplementary 4b).

GraphPad Prism 8 was used for all pre-clinical data analysis, utilizing two-tailed statistical tests with the assumption of normality. The application of two-way analysis of variance with Bonferroni correction was used to account for time and the different CAR cohorts. Flow cytometry data were analyzed using FlowJo v10.1.1, FCS Express v7.06.0015 and BD FACSuite v1.3. Transcriptomics data were analyzed using nSolver v2.0.134 and R studio 3.6. Clinical data are captured in the clinical database via the Encapsia electronic data capture (EDC) system v1.0. SAS 9.4 was used for clinical data analysis. Categorical variables are reported in terms of frequency and percentage, and continuous variables in terms of median and range unless otherwise specified. Time-to-event outcomes were summarized using the Kaplan–Meier method. Toxicity events are reported at the maximum grade experienced according to the CTCAE. The cellular kinetics parameters were estimated from the individual concentration versus time profiles using a non-compartmental approach in Phoenix WinNonlin.