AB0042 OPTIMAL CONDITIONS FOR THE DETECTION OF INFLAMMASOME ACTIVATION IN CD14+ CD16- MONOCYTES FROM HUMAN BLOOD BY FLOW CYTOMETRY

Abstract

BackgroundThe inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multimeric structure, known as the ASC speck. The aggregation of cytosolic ASC into ASC specks is therefore used as a readout parameter for inflammasome activity. The direct detection of ASC speck formation on a single cell basis through flow cytometry, can analyze inflammasome activated cells. This allows the investigation of inflammasome activity in a clinical setting.ObjectivesInvestigating the optimal conditions for a reliable identification of inflammasome activated ASC speck positive cells ex vivo using flow cytometry.MethodsFreshly donated blood from five different healthy donors was used for all experiments. The choice of anticoagulant, storage time and storage temperature were examined. PBMCs were isolated from blood collecting tubes with ethylenediaminetetraacetic acid (EDTA) or lithium heparin (LH). PBMCs were also isolated from LH blood stored at 4 °C, room temperature (RT), 37 °C and after different storage times. After isolation, PBMCs were fixed immediately and stained for flow cytometric analysis. ASC speck positive CD14+ CD16- monocytes and THP-1 cells can be generated through incubation with nigericin in PBS.ResultsWe adapted a method for flow cytometric analysis of ASC specks, with was previously described by Sester et al. [1]. As expected, we observed ASC speck formation after inflammasome activation, in CD14+ CD16-monocytes through a decrease in ASC fluorescent pulse width and an increase in ASC fluorescent pulse area. Monocytes in PBMCs collected from tubes with EDTA compared with LH showed significantly higher numbers of unspecific ASC speck positive cells. Blood storage at RT for 24 h can lead to an unspecific ASC speck formation. Storage at 37 °C resulted in contamination of the PBMC interface with erythrocytes, while blood stored at 4 °C resulted in severe cell clumping. Since storing LH blood for 24 h at RT lead to unspecific ASC speck formation in CD14+ CD16- cells, but not monocytes from freshly isolated PBMCs, the determination of the time until unspecific ASC speck signals occur was investigated. A significant increase in ASC speck positive CD14+CD16- monocytes was detected after 4 h storage at RT compared to directly processed samples and the number of ASC speck positive monocytes further accumulated over time. The incubation with nigericin in PBS leads to a significant increase in ASC speck positive CD14+ CD16- monocytes and THP-1 cells compared to incubation in RPMI media.ConclusionThe flow cytometric detection of ASC specks is adapted for practical clinical usability. To reduce background signals, LH- instead of EDTA blood collecting tubes are recommended. The LH blood should be processed within 2 h after blood collection and be stored at RT. To avoid nonspecific activation and formation of ASC specks, the PBMCs should be isolated directly after venipuncture and fixed immediately. It is also possible to freeze the PBMCs until further usage. However, this will cause some loss of ASC speck positive cells. With these settings, clinical samples can now be examined.