ADCC Capacity of PBMCs in CML Individuals

Raji cell line, which was provided by the existing collection of the Instituto de Salud Carlos III (Madrid, Spain), was used as a target to determine the antibody-dependent cellular cytotoxicity (ADCC) capacity of PBMCs isolated from CML individuals and healthy donors, as described previously [20 (link)]. Briefly, Raji cells were labeled with PKH67 green fluorescent cell linker (Merck KGaA, Darmstadt, Germany) and then coated with rituximab (50 μg/mL) (Selleckhem, Houston, TX, USA) for 4 h. Labeled rituximab-coated Raji cells were then co-cultured for 18 h with PBMCs (1:2 ratio). Early apoptosis of Raji cells was evaluated via staining with Annexin V conjugated with phycoerythrin (PE) (Immunostep, Salamanca, Spain). Data acquisition was performed with a BD LSRFortessa X-20 flow cytometer and data analyses were performed with FlowJo software v10 (Tree Star Inc.).