Lsfortessa

To investigate cellular uptake of nanoparticles in THP1 cell line, each of the three liposomal formulations was synthesized with addition of DOPE-NBD (fluorescent lipid/total lipid = 5/1000 mol/mol). Bare liposomes and liposome–protein complexes were administered to cells with serum-free medium. THP1 cells were plated at 500.000 cells mL⁻¹ in 12-well dishes, and then were incubated for 3 h with 10 µg mL⁻¹ of NBD-labeled liposomes in the Optimem medium. After the treatment, cells were washed with cold PBS and then run on a BD LSFORTESSA (BD Biosciences, San Jose, CA, USA). Cells were gated using forward versus side scatter to exclude debris. The data were analyzed using FlowJo software (FlowJo LLC data analysis software, Ashland, OR, USA) as elsewhere described^{63 (link)}.

After 72 h of co-culture, T and cancer cells were collected and pre-incubated with purified anti-mouse CD16/32 (TONBO, San Diego, USA) or Fc block (BD Biosciences, Franklin Lakes, NJ, USA) for 10 min at 20 °C before staining. The Transcription Factor Buffer Set (BD Biosciences, Franklin Lakes, NJ, USA) was used for intracellular staining. Antibodies used in flow cytometry experiments in this study are listed in Supplementary Table S1. When measuring the number of cells using polystyrene fluorospheres beads, a pre-determined number of beads was added to each pre-stained sample collected from the culture medium, and the number of cells after collection was calculated from the number of beads obtained using flow cytometry. CD45-positive cells were defined as T cells and all others as cancer cells. Flow cytometry was performed using a BD LSFortessa coupled with FACSDiva v6.1.3 and data analysed using FlowJo v10.7.1 software (all from BD Biosciences).