Annexin 5 fitc or apc

Cells were stained for 30 min (minutes) with the anti-human monoclonal antibody CD11b-PE (IM2581U, Beckman Coulter, Brea, CA, USA), CD14-FITC (555397, BD Biosciences, San Jose, CA, USA), or CD15-APC (551376, BD Biosciences, San Jose, CA, USA). Annexin V-FITC or APC (BD Biosciences, San Jose, CA, USA) and 7-AAD (Cell viability assay, BD Biosciences, San Jose, CA, USA) were used to assess cell viability. Data were collected on a MacsQuant cytometer (BD Biosciences, San Jose, CA, USA), and analyzed using the FlowJo software (BD Biosciences, San Jose, CA, USA).

Cytotoxicity assays were carried-out as follows: 50 µL aliquots of 3 × 10⁴ cells were seeded per well in 96-well plates and 50 µL aliquots of increasing concentrations of GRNLY or LUV-GRNLY were added and incubated for 24 h at 37 °C. For controls wells the same volume of KHE buffer was added. Cell death was analyzed using a FACScalibur flow cytometer (BD, Biosciences, Franklin Lakes, NJ, USA) after incubation with annexin-V-FITC or APC (BD Biosciences) in annexin binding buffer (140 mM NaCl, 2.5 mM CaCl₂, 10 mM HEPES/NaOH, pH 7.4) for 20 min. The 7-AAD staining (BD Biosciences) was also performed in some experiments. The data obtained was analyzed using FlowJo 0.7 (Tree star Inc., San Francisco, CA, USA).