Single-Cell Western technical library

In this Application Note, we show you how it may be possible to optimize the signal of poorly behaving antibodies with the Signal Enhancement Reagent on Milo.

In this application note, we validate Milo for the analysis of hiPSC-CM cultures. Further, we demonstrate how Single-Cell Western analysis can track phenotypic marker heterogeneity over time and monitor the relative proportion of cell subsets during culture differentiation

In this application note, we demonstrate how Milo can be used to identify and quantify neural subtypes in a heterogeneous neural sample, and monitor the differentiation of induced pluripotent stem cells (iPSCs) into neurons, astrocytes, and oligodendrocytes using R&D Systems research-grade or GMP differentiation reagents.

In this application note, we describe how you can generate great Single-Cell Western data with an Innoscan 710 scanner, which has the best combination of sensitivity, scan time and resolution needed to scan scWest chips.

In this application note, we demonstrate several measurements that are difficult or impossible to make with conventional westerns or flow cytometry. We uncover three distinct cell subpopulations that differ based on single- or co-expression of two different protein targets and that could not be distinguished using conventional westerns or flow cytometry. We also demonstrate Milo’s ability to detect discrete cell signaling states by measuring multiple phospho-proteins in individual cells within a population. Finally, we demonstrate how Milo can simultaneously quantify phospho- and total protein expression in each individual cell and quantify how the extent of phosphorylation varies across stimulated and unstimulated cell populations.

Every cell is unique, making cell-to-cell heterogeneity important in many areas of biomedicine including cancer pathogenesis, immuno-oncology and regenerative medicine. More and more high-profile publications are using single-cell analysis techniques to reveal variability in cellular response to a drug or stimulus. They're also uncovering variation in drug target expression within a tissue and identifying important subpopulations of cells within complex samples that play key roles in disease progression. Single-cell protein expression information is critical when you need to understand the fundamental composition and behavior of complex biological samples.

Milo, the first and only Single-Cell Western platform out there, lets you do Western blotting at the single-cell level. Now you can run Westerns on thousands of individual cells in parallel and get robust, Western-based information on protein expression heterogeneity in your cells. He also multiplexes so you can measure multiple proteins in each single cell. That means you can get a better understanding of correlations between target expression and characterize cell signaling in specific target-positive subpopulations of cells. Where your target is located in a cell doesn't matter to Milo — his fast, simple workflow lets you measure proteins both on and in each individual cell with the same workflow. It's easy to detect surface proteins and you don't have to worry about fixing and permeabilizing your sample to measure intracellular proteins. As an added bonus, Milo uses conventional Western antibodies which means you can measure diverse protein targets — even ones that don't have good flow cytometry antibodies. The best part? Scout™ Software automates your data analysis and gives you quantitative protein expression measurements in each single cell.

ProteinSimple’s analytical platforms give you the automation and scalability needed for the development and manufacturing of C> products, and with low volume requirements, we help you preserve these precious samples. Platform methods can be seamlessly transferred across labs and project phases, from discovery to manufacturing, giving you consistent results from start to finish.

Protein analysis plays an important role in stem cell research, and for most researchers, Western blotting is a key component of their toolbox. From verifying pluripotency and identifying lineage-specific cell types to identifying key modulators of cell signaling pathways, to evaluating disease models and therapeutic approaches, the right protein analysis
techniques can help you achieve success. In this eBook, we examine technological advances in Western blotting and protein analysis that are at the forefront of stem cell research. Learn how automated Western systems and Single-Cell Westerns can advance your stem cell research.

“With Milo, we observed a level of single-cell protein expression heterogeneity in our purified intestinal stem cell population that was not previously possible with conventional Western blotting.”

“Milo permits study of proteins at the single-cell level to understand the heterogeneity among metastatic cells within a population.”

Here, we provide instructions for updating the Milo Controller Software to Version 2.3.0

The tumor microenvironment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use an in-capillary immunoassay with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell Western to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by in-capillary immunoassay and single-cell Western. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, in-capillary immunoassay analysis identified mature populations by CD209 for DCs, a CD56+/CD3- phenotype for NK cells, and CD25 and Foxp3 expression for Tregs. Analysis of single cells provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like Western blot and flow cytometry.

See why leaders in regenerative medicine and cell therapy research use Simple Western™ and Single-Cell Western.

This Scientific Review highlights scientific articles that demonstrate how Simple Western and Single-Cell Western provide a holistic overview of signaling pathways in many different cell types, organisms, and environments, from the study of cancer to the impact of spaceflight.

Sickle cell disease results from a single point mutation in the β-subunit of hemoglobin (β-globin). Single-Cell Western is advancing sickle cell gene therapies.

InnoScan 710 Scanner Spec Sheet

Simple Plex assay for the detection of human B7 Homolog 1
(B7-H1) in culture supernatant (CCS), serum, plasma
(EDTA/Heparin), and urine

The Milo Single-Cell Western system can analyze multiplexed protein expression patterns in dissociated tissues to identify and quantify cell subtypes contained in heterogeneous tissues. Generating high quality single-cell suspensions from tissues is an important precursor to a Single-Cell Western experiment in order to preserve cell states and relative abundance in downstream analysis. We hope the following resources may be helpful when considering methods for dissociating your tissue of interest.

In this technical note, we demonstrate how diluting antibodies with Milk-Free Antibody Diluent when probing targets with a goat primary antibody delivers improved assay performance on the Milo Single-Cell Western platform.

This technical note will walk you through multiplexing with Milo and provide you with evidence of and guidance for efficient stripping to achieve multiplexing levels that match or exceed those of the most powerful multiplexed flow cytometers.