Molecular Pixelation (MPX™) deeply phenotypes immune cells by building spatial 3D maps of cell surface proteins in a highly multiplexed manner at nanoscale resolution. MPX yields millions of data points for protein colocalization, protein polarity, and protein abundance enabling novel discoveries. MPX is a sequencing based technology without imaging, and the product consists of a reagent kit and free software (i.e. there is no need for specialized equipment).

MPX, in comparison to genomics and transcriptomics techniques, yields abundance and spatial organization of surface proteins in 3D for single cells in suspension. MPX can study known protein interactions and reveal novel ones at the nanometer scale. MPX analyzes 84 proteins in parallel, for up to 1,000 cells per sample, whereas other technologies such as microscopy, only look at a few markers at a time for a limited number of cells, typically in 2D. Molecular Pixelation, therefore, enables a high multiplex,  spatial analysis of the cell surface proteome, for thousands of cells simultaneously, a new capability that was previously deemed impossible!

Yes, Molecular Pixelation has been published in Nature Methods. Please find the open access paper here: https://www.nature.com/articles/s41592-024-02268-9

The MPX product can be used to assess the abundance and spatial distribution of 84 proteins on the cell surface of human immune cells. The product consists of a reagent kit and an open-source software (i.e. there is no specialized equipment needed), and each kit contains reagents for 8 reactions in total.

The kits have 14-month shelf-life from the production date. The expiration date of your kit can be found on the front of each kit box.

Please store the different components at their recommended temperatures, as specified in the kit documentation.

You can request a quote on our website (https://shop.pixelgen.com/) or email hello@pixelgen.com and we will contact you.

We ship our product internationally and work with several distributors, please see https://www.pixelgen.com/about-pixelgen/distributors/ to learn if we use distributors in your region.

The first version of MPX v1 (product number PXGIMM001) was the initial product that Pixelgen launched. MPX v2 (product number PXGIMM002) is an updated product with an average of 2.2 times more data per cell, a 3.7 times higher average signal-to-noise ratio, and improved antibody performance. MPX v2 can also verify known protein interaction complexes, such as CD11a – CD18 (LFA-1), CD29-CD49D (VLA-4), and HLA-ABC – B2M. These improvements result from optimized chemistry, the updated panel “Immunology Panel 2, Human” as well as optimized algorithms for the spatial metrics found in the Pixelator software.

MPX uses barcoded antibodies, so called Antibody-Oligo Conjugates (AOCs), and DNA pixels to generate spatial 3D maps of the cell surface proteome. AOC binding introduces the abundance information of each target through a protein barcode, followed by two rounds of pixelation with DNA pixels to retrieve the spatial location of the proteins. MPX is a DNA-sequencing based method, without using any specialized equipment or imaging. Please see our Nature Methods paper to learn more details on the technology: https://www.nature.com/articles/s41592-024-02268-9

Molecular Pixelation is used to assess colocalization patterns of cell surface proteins, making it possible to study known protein interaction complexes (e.g. CD11a – CD18 (LFA-1), CD29-CD49D (VLA-4) and HLA-ABC – B2M), and discover novel protein-protein colocalizations.

MPX has been used for a variety of applications involving immune cells or cells expressing immunological targets (e.g. hematological cancer cells). These include drug development, hematology, immunotherapy, cell engineering and, basic biology, to mention some. Example studies could be assessing activation or exhaustion over a time course, comparing treated and control samples, analyzing the Mode of Action of drugs and drug targets, understanding response to treatment, etc.

No, MPX is developed for the analysis of cells in suspension. If you have access to fresh tumor tissue, MPX can be applied for cells dissociated from fresh tissue. Please contact support for guidance.

Please find the complete list of targets here: link. The 84-plex panel was designed to cover a variety of immune cell types and functions, including backbone targets as well as targets for activation, adhesion, exhaustion, differentiation, communication, etc.

The panel included in the kit is fixed, with targets that have been extensively validated. We have a target wish list where we are more than happy to take your feedback for potential future panels.

There are two types of controls in the AOC panel. The first part consists of mouse isotype control antibodies (mIgG1, mIgG2a, mIgG2b) that can be used to set non-specific binding levels. The other part contains an intracellular marker (AGBT) to validate that the cell membrane is intact and not permeabilized.

The Immunology Panel 2, Human (84 targets) is an updated panel with enhanced antibody performance. Targets CD127, CD163, CD197, CD137, and CD314 in Immunology Panel 1, Human were removed, and targets CD24, CD39, CD58, CD94, CD123, CD180, CD191, CD192, and CD335 were added in Immunology Panel 2, Human. The MPX User Manual remains largely the same,  although kit names and part numbers etc. have changed.

The Molecular Pixelation protocol is straightforward to perform. Training is typically carried out remotely, highlighting some general workflow tips and tricks. Please find a set of short Customer Support videos here: https://www.pixelgen.com/application-support/ to help guide you through some of the key parts of running MPX successfully. Our Support Team is ready to support you through all of the steps! 

MPX takes 2-3 days to run if running consecutively. The protocol contains several optional stopping points for workflow flexibility.

Cells can be either fresh or cryopreserved. MPX has been developed and optimized using PBMCs, but any cells that express immune markers can be used, e.g. PBMCs, isolated immune cell populations, blood cancer cells, cancer cell lines, and cells dissociated from fresh tumor tissue. Before MPX workflow, all samples are fixed with PFA.

The regular MPX protocol requires 500 000 – 1 000 000 live cells as input. However, we have developed a demonstrated protocol for handling low cell inputs, down to 50,000 live cells as input to fixation. Please contact support@pixelgen.com if you want to learn more. 

During the greater part of the protocol, automated cell counters, such as the Countess II Automated cell counter (ThermoFisher) can be used. The last step requires manual counting due to low cell concentrations, where we recommend glass hemocytometers or single-use counting hemocytometers. More details can be found in the User Manual.

We have assessed Trypsin + EDTA, TrypLE, and Accutase. Accutase was the mildest release treatment and impacted surface proteins minimally, and we therefore strongly recommend using Accutase for releasing cells. Trypsin + EDTA impacted the detection of certain proteins to a very large extent, with less but still impactful effects for TrypLE. 

No, the panel has been validated on the fixation protocol that is in the MPX User Manual and other fixation protocols could impact the antibody binding negatively and should not be used

If there is a need to use other PCR primers than the ones supplied with the kit, please contact support for guidance.

There is no need for additional instruments other than those available in a standard cell/molecular lab, such as a centrifuge and PCR machine.

We recommend a minimum of 200 000 reads per cell (including reads for PhiX). Since there are regions of low diversity in the reads, 15% PhiX is required. The minimum read configuration is R1: 44 and R2: 78, which means that e.g. 2×100, 2×150, 150 SE, etc. work well.

MPX has been validated for Illumina sequencers, with the usage of NextSeq2000, NovaSeq6000, and NovaSeqX+ sequencers.

The Pixelator software is compatible with 300 – 1000 cells output per reaction.

Please find recommendations here: https://software.pixelgen.com/pixelator/requirements and here: https://software.pixelgen.com/nf-core-pixelator/requirements

The NGS FASTQ files are imported to Pixelgen’s open source software Pixelator, undergoing several steps of quality control and analysis. The software uses the reads to produce graphs, ultimately generating one network of protein connections per cell. Pixelator has two main outputs, a Web QC Report for quality control metrics such as sequencing depth, as well as a PXL file. The PXL file should be used for downstream analysis to tailor the analysis to the specific scientific question. Please find more details here: https://software.pixelgen.com/

You need to be familiar with a programming language for data analysis such as Python or R. A basic understanding of analyzing single-cell data can be helpful, but not mandatory.

The polarity score of a protein will tell you if the spatial distribution of that protein is non-random. The more polarized a marker is in a cell, the higher the polarity score. For more details, please see https://software.pixelgen.com/pixelator/algorithms#mpx-polarity-scores.

The colocalization score of two markers will tell you if the two markers appear at the same locations more often than expected by random chance, as well as how colocalized they are. The more colocalized two markers are in a cell, the higher the colocalization score. The colocalization can also be used to build colocalization networks, to look at protein groups. For more details, please see here 

Yes. Pixelgen offers pre-computed layouts for each called cell, as well as tutorials for 3D visualization. Please find more information regarding cell visualization here: https://software.pixelgen.com/mpx-analysis/introduction

A component is a reconstruction of a cell, in a graph format consisting of nodes and edges.

Yes. Please see https://software.pixelgen.com/

We recommend that you run Pixelator through our nf-core/pixelator. For more information, please visit https://software.pixelgen.com/nf-core-pixelator/intro

Yes, we have tutorials based on both Python and R. Please see https://software.pixelgen.com/mpx-analysis/tutorials/introduction.

Yes. Please see https://software.pixelgen.com/datasets.