Facsaria 3 4l

Sigle cell suspension was prepared as described in the previous section. Next, a blocking step was performed with anti-mouse CD16/CD32 (Fc blocking) (Clone 93, Thermo Fisher Scientific) for 10 minutes, followed by incubation on ice for 30 minutes with the appropriate combination of fluorochrome-conjugated antibodies. Antibodies used for flow cytometry experiments are listed in Suppl. Table 6 (dilutions 1:300–1:600). Cells were analysed with the BD LSR Fortessa FACS (BD Biosciences) and sorted with FACS Aria III 4 L (BD Biosciences). Flow cytometry data including t-SNE analysis were performed using the FlowJo v.10 software.

Samples for gene expression profiling by RT-qPCR were set up in analogy to mRNA-seq experiments: for each condition a full 96-well plate with CD33- or CD123-directed CAR T-cells of the same healthy donor and MOLM13-TP53^+/+/MOLM13-TP53^−/− or MV4-11-TP53^+/+/MV4-11-TP53^−/− were incubated for 6 days. E:T ratios for respective co-cultures can be found in Appendix Table S3. After co-incubation, CAR T-cells and MOLM13/MV4-11-TP53 AML cells were sorted using a FACS Aria III 4L (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were sorted into FACS Buffer, spun down and pellets were stored at −80 °C until further processing. Total RNA isolation was carried out using the RNeasy kit (QIAGEN, Cat. No. #74104) according to the manufacturer’s instructions. Reverse transcription was done using SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (ThermoFisher, Cat. No. #11766500). qPCR was performed using TaqMan® Gene Expression Master Mix (ThermoFisher, Cat. No. #4369016) and TaqMan® Gene Expression Assays (HMGCR, Hs00168352_m1; HMGCS1, Hs00940429_m1; TCF7, Hs01556515_m1; EOMES, Hs00172872_m1; ACTB, Hs01060665_g1) on a 7900HT Fast-Real-Time PCR System (Applied Biosystems).

Lentiviral transduction of MOLM13-TP53 with Luciferase pCLX-UBI-GFP-Luc (kindly provided by Dr. Patrick Salmon, University of Geneva, Switzerland) was performed as described before (Myburgh et al, 2020 (link)) in the presence of hexadimethrine bromide (8 µg/ml, Sigma-Aldrich, St. Louis, MO, USA) by spin infection. Subsequent cell sorting of GFP+ populations on a FACS Aria III 4L (Becton Dickinson, Franklin Lakes, NJ, USA) of oligoclonal populations yielded pure MOLM13-TP53^+/+Luc⁺ and MOLM13-TP53^−/−Luc⁺ populations. The resulting clones were kept in culture and monitored for growth kinetics. Stable clones were selected and cryopreserved for future experiments.