Comparative Analysis of Methods to Reduce Activation Signature Gene Expression in PBMCs

Preserving the in vivo cell transcriptome is essential for accurate profiling, yet factors during cell isolation including time ex vivo and temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigate ex vivo activation signature gene (ASG) expression in peripheral blood mononuclear cells (PBMCs): transcription and translation inhibitors (TTis) and cold temperatures during isolation. Comparative analysis of PBMCs isolated with and without TTis revealed reduced ASG expression. However, TTi treatment impaired responsiveness to LPS stimulation in subsequent in vitro experiments. In contrast, cold isolation maintained experimental flexibility while similarly down-regulating ASG expression compared to TTis. Notably, addition of TTis during cold isolation offered minimal additional advantages. Thus, while both TTis and cold isolation effectively reduced ASG expression, we found cold isolation to be a more practical approach. Overall design: To study activation signature gene mitigating protocols for blood mononucleated cells (PBMCs), we isolated and treated PBMCs in different experimental conditions. Gene expression profiling was performed from patient-derived peripheral blood mononucleated cells (PBMCs) to compare two activation signature gene-mitigating cell isolation protocols; addition of transcription and translation inhibitors or cold temperature. Transcriptomic signatures were also compared from time-point thaw samples from PBMCs isolation using a standard protocol at room temperature and incubated in vitro for 0, 2, or 24 hours.