L1825 bc

Tumor tissues were obtained within 1 to 2 h after surgical removal, washed in sterile Dulbecco’s phosphate-buffered saline (PBS) (L1825-BC – Merck Millipore, Italy), and mechanically minced into small pieces (2 to 4 mm). Minced samples were digested using a tumor dissociation kit in a disposable gentle MACS™ C-Tube (Miltenyi Biotec, Italy) according to the manufacturer’s instructions. Samples were digested for 60 min at 37°C in a gentle MACS Octo dissociator, filtered through 70-μm sterile cell strainers, centrifuged at 300 × g for 5 min, and resuspended in a DMEM and HAM’S F12 media mixture (2:1) (Gibco) containing 50 IU/mL penicillin-streptomycin and 4 mM glutamine. Viable cells were counted using an optic phase-contrast microscope [7 (link)].

Freshly obtained tumor tissues (within 1–2 h after surgical removal) were washed in sterile Dulbecco's phosphate-buffered saline (PBS) (L1825-BC—Merck Millipore) and mechanically minced into small pieces (2–4 mm). Minced samples were digested using a tumor dissociation kit in a disposable gentle MACS™ C-Tube (Miltenyi) according to the manufacturer's instructions. Samples were digested for 60 min at 37°C in a gentle MACS Octo dissociator and filtered through 70-μm sterile cell strainers, centrifuged at 300 × g for 5 min, and resuspended in a mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F12 media (2:1) (Gibco) containing 50 IU/ml penicillin–streptomycin and 4 mM glutamine. Finally, viable cells were counted using an optic phase contrast microscope.

Surgical specimens were retrieved 1 to 2 h after surgery, washed in 50 mL sterile Falcon with Dulbecco’s phosphate-buffered saline (D-PBS) (L1825-BC—Merck Millipore, Italy), and mechanically minced into small pieces (2 mm to 4 mm). Samples were digested for 60 min at 37 °C in a gentle MACS Octo dissociator according to the manufacturer’s instructions, with Milteny tumor dissociation in a MACS™ C-Tube (Miltenyi Biotec, Italy). The cell suspension was then filtered through 70-μm sterile cell strainers, centrifuged at 300 × g for 5 min, and resuspended in a DMEM and HAM’S F12 media mixture (2:1) (Gibco) containing 50 IU/mL penicillin-streptomycin and 4 mM glutamine. Viable cells were counted using an optic phase-contrast microscope.