Aims:

Intracellular staining and flow cytometric analysis of lymphocytes is commonly used to assess cytokine production at the single-cell level in heterogeneous samples. Here, we describe a novel fluorescence in situ hybridization (FISH)-based flow cytometry assay (PrimeFlow RNA Assay) used in combination with intracellular antibody staining to study the kinetics of the transcription and translation of IFN-gamma and TNF-alpha in lymphocytes.

Methods:

Normal human peripheral blood mononuclear cells were stimulated with PMA and Ionomycin for 0-5 hours. Cells were fixed, permeabilized, and intracellularly stained with antibodies for CD8, IFN-gamma, and TNF-alpha. Next, cells underwent a series of hybridization steps to label mRNA for IFN-gamma and TNF-alpha. Data were collected on an LSRFortessa and analyzed using FlowJo software.

Results:

IFN-gamma mRNA was upregulated in CD8+ and CD8- lymphocytes within 1 hour after stimulation, while protein levels were not detected until 2 hours, after which both mRNA and protein were maintained for the next 3-4 hours. In contrast, TNF-alpha mRNA and protein were both upregulated within 1 hour after stimulation and expression was maintained in CD8+ cells while expression in CD8- cells peaked between 1-2 hours and then decreased over the next 4 hours, with the decrease in mRNA preceding the decrease in protein.

Conclusions

Using the PrimeFlow RNA Assay, we found that induction of IFN-gamma and TNF-alpha mRNA and protein exhibit unique kinetics and that TNF-alpha protein and mRNA are differentially regulated in CD8+ and CD8- lymphocytes. This new PrimeFlow RNA Assay enables the study of gene expression at the single-cell level in heterogeneous samples without the need for sorting specific subsets and the ability to compare and contrast the kinetics of mRNA and protein induction.