Flow cytometry method to identify arginase-1 expressing MDSCs.

Myeloid-derived suppressor cells (MDSCs) contribute to immune suppression in cancer through arginase-1 (ARG1), which limits T-cell activation. Conventional assays such as ELISA or qPCR cannot determine the cellular source of ARG1 in blood. Recognizing the importance of ARG1 in immunosuppression, we developed and validated a standardized flow cytometry method using a commercially available ARG1 antibody to measure intracellular ARG1 in MDSC subsets. This method includes detailed instructions, including experimental optimization using positive and negative controls, to help other researchers perform flow cytometric measurement of ARG1 expressing cells. In this method, peripheral blood mononuclear cells were stained for lymphoid and myeloid markers (CD3, CD20, CD56, HLA-DR, CD33, CD11b, CD14, and CD15), fixed, and permeabilized before intracellular ARG1 staining. Antibody specificity was verified using neutrophils as positive and CD4⁺ T cells as negative controls. The method was applied to blood samples from patients with bladder cancer (n = 23) and healthy volunteers (n = 10), demonstrating reliable detection of intracellular ARG1 in MDSC subpopulations. This accessible protocol provides an adaptable method for quantifying cell-specific ARG1 expression and studying MDSC-mediated immunosuppression.•The flow cytometry method described here was developed by validating the arginase1 antibody binding specificity using cells expressing high and very low levels of arginase-1.•Arginase-1-expressing cells of the peripheral blood monocytes were identified using this method.•This method can be adapted to any cell type and will be a valuable tool in future immunosuppression studies.