Schoon, Hannah

BACKGROUND Cytometry by time‐of‐flight (CyTOF) or mass cytometry is a form of flow cytometry. CyTOF uses antibodies bound to metal isotopes as tags instead of fluorochromes normally used in standard flow cytometry. These nonradioactive isotopes of heavy metals allow mass cytometry to analyze up to 50 different parameters without significant signal overlap. Due to the accuracy of mass cytometry, researches are able to investigate multiple cell markers at once, which is not possible with standard fluorescent flow cytometry (FFC). CyTOF works by feeding a cell sample tagged with conjugated antibodies into a nebulizer where cells are aerosolized. The aerosolized samples pass through an argon plasma torch and become an ionized atom cloud. This cloud passes through a quadrupole where low mass ions are filtered out resulting in a cloud enriched for the heavy metal reporter tags. The tags enter the time‐of‐flight chamber and further separated by mass as they reach the detector AIMS The main aim of this project was to optimize the stimulation conditions that are best suited for use with a CyTOF T cell functional panel. This would provide a method for decreased cell loss in sample preparation. METHODS T‐cell‐enriched samples underwent stimulation using 4 different condition: 4‐hour stimulation of PMA/Ionomycin, 1:1 ratio of Dynabeads at 24h, 48h, and 72h. Brefeldin A was placed in every well containing cells to act as transport inhibitor. FFC was used to test cell recovery and cell death between stimulation techniques and stimulation strength was observed using CyTOF. RESULTS There was no significant difference in 4h unstimulated cells and 24h unstimulated cells in both cell recovery and cell viability. However, comparing the PMA/Ionomycin unstimulated cells to the 4h stimulated cells did produce a significant decrease in cell recovery and cell death. Because this technique is a non‐physiological stimulation, this result was expected. The use of FFC also allowed us to see the significant loss of cells in recovery and death after 24h stimulation with the 48h and 72h Dynabead assays. The “sledge hammer” effect is clearly visible with the PMA/Ionomycin with 2.73% of IL‐4 cells being stimulated compared to only 0.28% in the 24h Dynabead sample. This trend of PMA/Ionomycin producing a strong stimulation signal was a trend. CONCLUSIONS We determined that T cell culture with Brefeldin A beyond 24 h significantly decreases cell viability and recovery. In observing the stimulation for Dynabeads at 24h, the cells are not directly affected while PMA/Ionomycin does cause a significant loss in cell viability and total cells recovered. PMA/Ionomycin is capable of stimulating the production of all measurable cytokines, even rare populations such as IL‐10‐ or IL‐6‐producing T cells. Dynabeads resemble a more physiological stimulation condition and is able to activate all T cell subsets except IL‐6‐producing T cells. Support or Funding Information The Berner Charitable and Scholarship Foundation, Funded by the Immune Monitoring Core and The Division of Hematology This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .