Cd8 apc vio770

All animals were cared for in a specific pathogen-free room and treated following the animal care protocol approved by an animal committee. Mouse LLC1 (1 × 10⁶) cells were subcutaneously injected into 8-week-old -C57BL/6 mice. After one week of inoculation, the mice were intraperitoneally administered melatonin (30 mg/kg, three times per week) for 4 weeks. The tumor volume and mice body weights were monitored every 2 days during the experiments. Tumor volume was calculated using the following equation: length × (width)2 × 0.5. To analyze tumor-infiltrating lymphocytes (TILs), freshly isolated tumor tissue was cut into small pieces and disassociated by the gentleMACS tumor dissociation kit (Miltenyi Biotec). The suspension was further treated with a RBC lysis buffer to remove red blood cells. Approximate 1 × 10⁶ isolated cells were incubated with fluorophore-conjugated antibodies including CD45-APC, CD11b-PE, CD3-FITC, CD8-APC-Vio770, Ly6G-FITC, CD4-PE-Vio770, and F4/80-APC (Miltenyi Biotec) and analyzed by the BD FACSVia flow cytometer (BD Biosciences) [43 (link)].

Large-scale manufacturing of CAR-T cells on CliniMACs Prodigy was carried out under GMP-like conditions into Gene-Cell Therapy clean rooms of the Cell Therapy Unit of Hospital Universitario Reina Sofía (Córdoba, Spain). Two different aphereses from a healthy donor were thawed, and around 100 × 10⁶ (link) T cells were inoculated into the CliniMACs prodigy bioreactor (Miltenyi Biotec). CD4 and CD8 cells were selected with CD8 and CD4 Reagent (Miltenyi Biotec), cultured with IL-7 and IL-15 (Miltenyi Biotec), and activated with αCD3 andαCD28 GMP T cell TransAct (Miltenyi Biotec). On day 2 of the process, these cells were transduced with AWARI-LVs (MOI = 5). Cells were cultured in TexMACs GMP medium containing GMP-grade IL-15 and IL-7 (Miltenyi Biotec) for 9 or 10 days. Final product was collected with 100 mL of NaCl 0.9% + 0.5% human serum albumin (HSA).
Cells were stained with CD3-APC, CD4-FITC, CD8-APC Vio770, CD14-PE Vio770, and CD45-Vioblue (Miltenyi Biotec). To assess the efficiency of transduction, CAR-T cells were stained with CD-19 Biotin and anti-Biotin PE (Miltenyi Biotec). Viability was tested by using 7-AAD (Miltenyi Biotec). Phenotype was determined with CD45RA-APC (HI100) and CCR7-BV421 (2-L1-A RUO) from BD Pharmingen. Cells were acquired on a MACsQuant cytometer (Miltenyi Biotec) and analyzed with MACsQuantify Analysis Software (Miltenyi Biotec).