Introducing the Dish Soap Protocol: A Unified Approach for Multi-Modal Intracellular Staining

Abstract

Recent advances in dyes and cytometers have seen an exponential increase in the ability to perform multidimensional flow cytometry. As we increase our capacity to extract information from cells, the need to acquire different types of data simultaneously from the same cells becomes limited by fixation buffer compatibility. Accessing the intracellular compartments for staining, particularly with large molecular weight structures such as antibody–fluorophore conjugates, requires a balance between solubilizing lipids to create entry points through the membranes, while using crosslinking fixatives to maintain structural integrity of the cell and prevent the loss of intracellular contents. Unfortunately, common fixation protocols result in a trade-off, where preservation of fluorescent proteins and accessibility of nuclear staining are often diametrically opposed. In this article, we detail the impact of various fix-perm reagents on key features, such as nuclear staining, green fluorescent protein (GFP) retention, intracellular cytokine staining, epitope retention, scatter profiles, cell recovery, and fluorophore stability. We provide a protocol for the use of “Burton's Best Buffer”, which is readily made at 100-fold lower cost than commercial buffers, which can be used to overcome the limitations of current fixation buffers and achieve simultaneous efficient detection of transcription factors, cytokines, and endogenous fluorescent proteins, among other uses. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Dish soap protocol