Population Representation

The Innovative LeviCellTM Platform Removes Debris and Dead Cells While Maintaining Original Representation of Different Cellular Populations.

Introduction

Cellular researchers hold three sample prep parameters as critically important to downstream analyses: cell viability, the absence of debris and aggregates, and the preservation of original cell population representation. However, scientists are often plagued with one or more of the following challenges: low viability, insufficient debris removal, large aggregates, or low purity.  Consequently, replicability and reproducibility are compromised, hindering the collection of data that may potentially produce revolutionary results.

The LeviCellTM is a ground-breaking levitation platform that uses less than 1 psi of pressure to gently separate cells, providing researchers the ability to optimize cell viability and enable reproducibility – a key component of successful assays. In addition, the LeviCellTM’s touch-free, label-free, three-step, 20-minute process minimizes aggregate formation, maximizes debris removal, and preserves original cell population representation. This increased efficiency in sample preparation translates to more robust results in downstream applications.

Methods

The LeviCellTM gentle cell separation process enables retention of cell health and through highly efficient enrichment produces maximum sample viability, while preserving cell population representation for downstream assays.  The simple three-step LeviCellTM process streamlines the workflow, simplifies the enrichment process, and minimizes the common issue of low viability and yield due to contamination and damage from multiple instances of staining, handling, and washing.

Live cell populations levitate much higher than dead cells and debris, thus allowing for clear separation and collection by the LeviCellTM, as shown.  Please note that the fluorescent dyes used in the provided image were used solely to highlight the separation process and are not required to use the LeviCellTM.

Results

In order to validate the performance of the LeviCellTM system, PBMCs were re-suspended in levitation reagent and enriched for live cells using the LeviCellTM.A portion of the sample was set aside. Both live and dead channel outputs and the unsorted sample were stained with PI and analyzed by flow cytometry on a Sony®SH800S cytometer.

The live channel, as indicated by the graph in the middle, shows nearly threefold enrichment (87% viability) compared to the unsorted sample (Pre-LeviCellTM Sort), which shows 31% viability. The dead channel, as signified by the graph on the right, contains 96% dead cells. This clearly demonstrates the LeviCellTM platform’s ability to maximize viability and debris removal.

No negative effects on population representation

To demonstrate the LeviCellTM’s gentle process – which enables robust yields without affecting population representation of the original sample – PBMCs were suspended in Levitation AgentTM and enriched for live cells using the LeviCellTM. A portion of the original sample was analyzed independently. Both live and dead channel outputs as well as the unsorted sample were blocked with TruStain human Fc Block for 15 minutes at RT then stained with 5 μLeach of anti-CD45 (PE), anti-CD3 (FITC), anti-CD11b (APC), and 10 μL of anti-CD19 (PerCP-Cy5.5) for 1 hour on ice. Samples were then washed with a FACS buffer (0.5% BSA in PBS) once prior to analysis on a Sony SH800S cytometer.

The live channel output (red bar) shows a similar amount of CD3+, CD19+, and CD11b+ cells within the CD45+ lymphocyte population as was present prior to sorting (blue bar).  The consistency of robust live cell yield across all cell types is testament to how the LeviCellTM platform’s fast, gentle, and powerful separation technology enables the preservation of original population representation.

No effects on activation state post-processing

The LeviCellTM’s ability to sort without affecting activation states was tested using notoriously sensitive macrophages. Macrophages can be polarized into two main states: M1 (pro-inflammatory) and M2 (anti-inflammatory). The J774 mouse macrophage cell line (cultured in DMEM buffered with 10% FBS) was treated with IFN-𝞬 (50ng/mL) and IL-4 (20ng/mL) respectively for 24 hours. Untreated macrophages (M0) were kept as control. The starting viability of the J774 cells going into the LeviCellTM was 20% for each polarization state.

Enrichment to 88 – 100% viability was observed across three different runs post-sort. Following enrichment, qRT-PCR was performed to examine the expression of genes indicative of the M1 and M2 states in these cells to show that their activation states were not compromised by the LeviCellTM sort.

No significant differences in gene expression were observed between the input and output cells for each activation state. This shows that the LeviCellTM sort is gentle enough to sort sensitive cells such as macrophages without affecting their polarization states.

Conclusion

Reproducibility of results  are paramount to the success of an assay and its contribution to the advancement of science. In cellular research, downstream applications are heavily dependent on sample preparation results: high viability and yield, low debris and aggregates, and population representation conservation of all cell types within the sample.

Consistency is  a key factor that helps determine the outcome of downstream assays.  The LeviCellTM platform has consistently demonstrated its ability to produce robust yields and viabilities of all cell types without affecting the original sample population representation or activation state, enabling a strong starting point for downstream applications. As well, LeviCellTM‘s thorough removal of debris optimizes the purity of the sample, facilitating accurate downstream analyses – ultimately improving the reproducibility of your assay.