Establishment of a flow cytometry-based test for the detection of pyroptosis of BMDM

Objective To establish a flow cytometry-based test for the detection of pyroptosis of murine bone marrow-derived macrophages (BMDM). Methods Bone marrow cells were isolated from wild type (WT) C57BL/6 mice and/or caspase-1-/- C57BL/6 mice and then stimulated with macrophage colony-stimulating factor (M-CSF) to differentiate into murine BMDM. PBS, LPS and LPS+ adenosine triphosphate (ATP) were respectively used to stimulate the BMDM. Western blot assay was performed to detect the cleavage of IL-1β and caspase-1. The levels of IL-1β in the supernatants of cell culture were measured by ELISA. Lactate dehydrogenase (LDH) released in the culture media was detected by using LDH kit. The pyroptosis of murine BMDM was detected by using flow cytometry analysis after double-staining with TMR red+ caspase-1, AnnexinⅤ+ caspase-1 and propidium iodide (PI)+ caspase-1. Results IL-1β was detected in the culture medium of BMDM treated with LPS+ ATP and the cleavage of IL-1β and caspase-1 was confirmed by Western blot assay, which indicated that the NLRP3 inflammasome was activated by LPS+ ATP treatment. Compared with the caspase-1-/- mice group, higher levels of LDH were detected in the culture medium of BMDM isolated form the WT mice. Results of the flow cytometry analysis after staining BMDM with caspase-1 plus AnnexinⅤ or PI showed that more cells undergoing pyroptosis were detected in the LPS+ ATP treatment group than that in LPS or PBS treatment group, which were consistent with the results of the reported flow cytometry with caspase-1+ TMR red staining. Conclusion The flow cytometry-based test with double-staining of caspase-1 plus AnnexinⅤ or PI could be used for the detection of pyroptosis of murine BMDM. Key words: NLRP3 inflammasome; Pyroptosis; Flow cytometry