Fast, Simple, and Label-Free Nuclei Enrichment
Nuclei have quickly become one of the main targets for single-cell research and development when working with difficult to process tissues and samples. While working with cells should be much simpler and more direct than nuclei, the realities of processing tissues are quite often much more complex and difficult than expected.
Tissues vary in their cellular composition, extracellular matrix, and dissociation behavior when processed. As such researchers have found it increasingly difficult to establish well-validated and uniformly performing dissociation and purification protocols that produce a reliable, high-quality single-cell suspension.
Alternatively, nuclei workflows overcome many of these challenges by not only providing a much more uniform and standardized starting material for single-cell analysis but also remove some of the potentials for gene expression changes associated with tissue dissociation and processing.
Unfortunately, nuclei preparation comes with its own challenges. Specifically, how to purify extracted nuclei away from dead cells and debris. Traditional methods continue to be difficult and time-consuming to use, producing poor yields, and purities.
The LeviCellTM offers a fast, simple, and highly efficient method to purify nuclei.
Nuclei were isolated from flash-frozen lung and brain tissue by incubating with Nuclei Lysis Buffer (ThermoFisher) for 30 minutes on ice. Nuclei were then washed with PBS containing 0.1% BSA two times before resuspending in Nuclei Storage Buffer (1M Sucrose + some stuff). Nuclei were prepared for separation by resuspending 3e5 nuclei in Levitation Buffer containing 100mM Levitation Agent. A sample was set aside for comparison after separation. Nuclei were separated in the LeviCellTM using a 30 minute equilibration period. The outputs were stained with PI and imaged using an Echo microscope.
For quantitative analysis of nuclei enrichment, Jurkat nuclei were isolated as above. Aliquots from before and after sorting were stained with PI and Cellbrite Fix (Green), a membrane dye whose signal is dependent on the amount of membrane present. After 15 minutes at room temperature, the samples were analyzed by flow cytometry on a Sony SH800S Cell Sorter.
The nuclei stain brightly with PI while cellular debris does not. Images of the stained inputs and outputs from the LeviCellTMl show a significant reduction in the amount of unstained debris for both tissue samples. The lung tissue produced more debris than the brain tissue (Figure 1A and C) but both samples show marked reduction in debris after sorting with the LeviCellTM (Figure 1B and D).
Figure 1. Microscope images of unsorted (A, C) and sorted (B, D) nuclei derived from lung (A, B) and brain (C, D) tissue. Samples were stained with PI and imaged at 20x. Objects stained with PI are nuclei while unstained objects are designated debris. There is much less debris in the sample after sorting with the LeviCellTM.
For quantitative analysis of nuclei enrichment, sorted and unsorted samples were stained with PI and Cellbrite Fix and analyzed by flow cytometry. The debris does not stain for PI but does stain with Cellbrite Fix depending on the size of the debris. The dead cells and nuclei both stain with PI but the dead cells, which contain more membrane, show a higher Cellbrite Fix signal than the nuclei. After sorting both the debris and dead cell populations are reduced while the nuclei population is enriched from 28.28% to 43.98%.