Dna macsfectin

Lentiviral vector production was performed as previously described 51 (link). In brief, HEK293T cells at a confluency of 70-90% were transfected with DNA:MACSfectin™ (Miltenyi Biotec) complexes using a total amount of 50µ DNA per T175 flask. HEK293T cells were then incubated overnight and sodium butyrate was added at a final concentration of 10 mM. Lentiviral vector containing supernatant was collected 48-60 h after transfection and following a filtration through 0.45 μm-pore-size PVDF filters subjected to centrifugation at 4 °C and 4,000×g for 24 h. Air-dried pellets containing lentiviral particles were resuspended at a 200-fold concentration with 4 °C cold PBS, aliquoted and stored at -80 °C.

To generate VSV-G pseudotyped lentiviral particles encoding the CAR of interest, lentiviral vector production was conducted as previously described by Schäfer et al. [67 (link)]. Briefly, HEK293T (ATCC, Manassas, VA, USA) cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS, Catus Biotech, Tutzing, Germany) and 100 µg/mL Primocin (InvivoGen, San Diego, CA, USA) to a confluency of 70–90%. Transfection was performed with a 1:2 DNA:MACSfectin™ (Miltenyi Biotec, Bergisch Gladbach, Germany) mixture with a total amount of 50 µg DNA per 175 cm² flask. Sodium butyrate (Merck, Darmstadt, Germany) was added 20 h after transfection to a final concentration of 10 mM, and the lentiviral supernatant was harvested 48 h after transfection. Following a spinoculation for 16 to 20 h at 3500× g and 4 °C, virion-containing pellets were resuspended at a 200-fold concentration in TexMACS™ Medium (Miltenyi Biotec) and stored at −80 °C until further usage.