Do we have any recommendations for profiling peripheral blood mononuclear cells (PBMCs) with Teton™?

PBMCs are a heterogeneous mixture of peripheral blood cells, primarily lymphocytes (T, B, and NK cells), monocytes, and dendritic cells. Lymphocytes make up the majority of PBMC composition and are typically <200 μm2 in area (for resting cells). HeLa cells, which are Element’s default cell line for Teton Cytoprofiling run review, are ~1,600 μm2 on average in comparison. As a result of this significantly smaller size, high intracellular polony density can impact barcode assignment efficiency and overall run performance. Below are setup recommendations and performance metrics for PBMC runs using Element Bioscience’s Teton or Teton Atlas™ Cytoprofiling kits. Note that PBMCs have not been tested on Teton Atlas and are not recommended with Teton Atlas High Output.

Teton Cytoprofiling - Experimental Set-up with PBMCs

We recommend resuspending cells at 1,000,000 cells/ml resulting in a seeding density of ~150,000 cells per well or ~30% well confluency for a 12-well slide kit. For sample preparation, it is recommended to follow the guidance for attaching and fixing suspension cells in the Teton Cytoprofiling User Guide.

It is strongly recommended to optimize cell seeding prior to your PBMC Cytoprofiling run. Reference the Cell Culture Hub for guidance on seeding density titration in a plate-based format prior to using one of Element’s supported optimization kits. More details can be found in the Teton Optimization and Screening User Guide.

A PBMC segmentation model is available on instrument by inputting PBMC as a cell type when setting up the run manifest. Additionally, the PBMC Segmentation Model can be found in Element’s GitHub repository for use with off-instrument segmentation.

Teton Cytoprofiling Run Metrics for PBMCs

An Element-generated run using the Teton Human Immuno Panel Kit and 3 Focus panels (Cytokine Signaling, T Cell Activation, and Innate Immunity) with the Teton 12-Well PLL Slide Kit generated the following data.

Cells were treated with phytohemagglutinin (PHA) and fixed with 4% formaldehyde as described in the user guide. These cells were seeded at 5-6% confluency with ~30,000 cells/well due to limited available PBMCs. <20% confluency is not recommended and may affect assignment rate though clustering, counts per cell, and spatial resolution should largely remain unaffected by this. In this run review, the results are compared to the read quality metrics as described in the Run Review Guide for Cytoprofiling. HeLa cells are the default cell type that was used to generate these run review metrics.

Average Assigned Counts per Cell

Assigned counts per cell is defined as the average number of transcripts and proteins assigned to a known barcode within a segmented cell.

On average, ~105 counts were detected per cell, with ~40 of these coming from protein and ~65 coming from RNA. HeLa cells, by comparison, typically produce 2,000-2,500 counts/cell. This discrepancy is mainly due to the smaller size of PBMCs, but the average cell assigned counts per mm2 was also slightly lower.

Average Cell Assigned Counts per mm²

The Average Cell Assigned Counts per mm2 is the number of assigned polony counts per mm2 of segmented cell data.

The average cell assigned counts per mm2 for PBMCs was ~1,200 k /mm2 across all batches. HeLa cells, for reference, typically produce 1,400-1,700 k / mm2. The average cell assigned counts per mm2 per batch ranged from 101 k/mm2 - 180k/mm2 for the RNA targets, and 279k/mm2 - 342k/mm2 for protein targets. This is within the target threshold of 100 k – 500k / mm2 but is closer to the lower end of the range, as apparent from the slightly lower density relative to HeLa. This is largely due to the lower assignment rate.

Average assigned count/mm² for eight batches from two different wells of PBMCs.

Assigned Rate

The assigned rate is the percentage of total polonies that are assigned to a protein or RNA target barcode, per well and per batch.

For PBMCs, the assigned rate varied between 45-80% depending on the well and batch but averaged ~58%. As per the run review guide, assignment rate is considered a warning below 65% and an error below 50%. This is in part due to the high intracellular polony density, making it difficult to discern clean avidite signals, and the lower confluency.

Assigned rate of two different wells of PBMCs and eight batches per well.

Extracellular Ratio

The extracellular ratio is the percentage of assigned polonies located outside cell segmentation boundaries, per well.

For PBMCs, the extracellular polonies ratio was <3% for RNA targets and <7% for protein targets (Figure A). The desired extracellular polonies ratio is below 25%, so this PBMC data is well within the desired specification.

Extracellular ratio of two different wells of PBMCs and eight batches per well.

Spatial Resolution

The cell membrane is shown in green, actin in red, and nucleus in blue. Due to the small size of PBMCs, limited morphological resolution is available as seen in the above tile image. Despite this, cell segmentation is still robust based on visual inspection of cell boundaries.

Cell Clustering Data

Creating UMAP clusters and visualizations depends on how diverse and wide-ranging the input data is, as well as on the settings used for the algorithm (such as the resolution). Here, 6 distinct clusters have been identified using canonical immune markers found within the Teton Human Immuno Panel Kit (830-00039) in addition to the custom protein panels Teton Focus Protein Panel, Set 1, Cytokine Signaling (830-00046) and Teton Focus Protein Panel, Set 2, T Cell Activation (830-00047) , shown as overlays across the UMAP. Here, 6 distinct clusters have been identified using canonical immune markers found within the Teton Human Immuno Panel Kit (830-00039) in addition to the custom protein panels Teton Focus Protein Panel, Set 1, Cytokine Signaling (830-00046) and Teton Focus Protein Panel, Set 2, T Cell Activation (830-00047)

Possible cell types based on representative markers detected

What are the benefits of running PBMCs on Teton compared to scRNAseq?

Teton enables very high throughput, at >1.5M PBMCs per run (depending on assay conditions and slide kit format), in a simple library prep-free workflow with no downstream sequencing required. With just 45-minutes of hands-on time, this results in a cost-effective, efficient workflow with added protein and morphology resolution. However, morphological resolution is limited compared to larger cells and the clustering resolution may be reduced due to lower feature counts per cell. Thus, experiments that require high throughput (e.g., rare cell detection), low workflow complexity, or multiomic profiling may be good candidates for Teton profiling.

For other information on cell types and set-up guidelines for Teton Cytoprofiling kits, please see What types of cells can I run with Teton™ CytoProfiling Kits? focused on various cell types that have been optimized with the Teton kits. Additional details about PBMC setup or any other newly tested cell types will be included on this page.