Facscanto ow cytometer

The cells were resuspended in 1 ml of precooled PBS. The number of cells in each tube was adjusted to 1×10 6 . Surface staining was performed using PE-conjugated anti-mouse CD120 (Biolegend, CA, USA, 113003) and Brilliant Violet 421 TM -conjugated anti-mouse CD206 (Biolegend, CA, USA, 141717) antibodies. For intracellular staining, the cells were xed with 2% paraformaldehyde (Solarbio, Beijing, China) and lysed with 1× Intracellular Staining Perm Wash Buffer (Biolegend, CA, USA). Then, the cells were incubated with PE-conjugated iNOS (Biolegend, CA, USA, 696805) and APC-conjugated Arg-1 (R&D, MN, USA, IC5868A) antibodies. All samples were evaluated with a BD FACSC Anto ow cytometer, and the data were analyzed with CytExpert software.

MRD testing was performed at the 3rd month after the end of rst-line treatment. The bone marrow MRD assessment method used in this analysis was described previously (Gu et al. 2018 (link)). EDTA-anticoagulated bone marrow aspirate (2 mL) was washed with PBS three times, and then the cell density was adjusted to ≤ 20×10 6 /ml. Samples (100 µL) were then incubated with the following antibodies: anti-CD38, anti-CD56, anti-CD19, anti-CD20, anti-CD54, anti-CD138, anti-CD45, anti-cytoplasmic κ, and anti-cytoplasmic λ antibodies. One to two million nucleated cells were analysed using a BD FACSCanto ow cytometer. Cells were gated based on combinations of the markers CD38, CD138, and CD45 and SSC, and at least 50 plasma cells were analysed. Abnormal plasma cells were differentiated from normal plasma cells by surface markers and cytoplasmic expression of light chain. The results were positive if ≥ 20 plasma cells with an abnormal phenotype were detected. The sensitivity of our ow cytometry detection of MRD was 5*10 - 5 to 10 - 5 cells.