Milo

Single-Cell Westerns

Milo™ is the world's first Single-Cell Western platform. He measures protein expression in thousands of single cells in a single run so you can profile heterogeneity in your samples. Just load your cell suspension and the scWest chip captures ~1,000 single-cells. Milo then does a fast, 1 minute SDS-PAGE separation on each single-cell lysate on-chip. Then just probe with your favorite conventional Western antibodies to measure ~12 proteins per cell using a variety of multiplexing strategies. Use Single-Cell Westerns to unlock the single-cell proteome and measure more of the proteome than is possible with any other single-cell technique.

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How Can Milo Help You?

I need to profile heterogeneity in complex samples

Milo measures protein expression in ~1,000 single-cells in one 4 hour experiment so you can quantify expression heterogeneity of your target and the percentage of cells in your sample that are target positive.

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Milo chart - heterogeneity - 51% KRT8+ cells

I need protein validation of my single-cell RNA-seq data

Use conventional western antibodies to validate your single-cell RNA-seq data with single-cell protein data. Plus, scWest chips can be archived for up to 9 months after you run them so you have plenty of time to get your sequencing results back before you have to probe for your targets of interest.

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Milo chart - Protein validation of my single-cell RNA-seq data

I need multiplexing to detect multiple proteins at the single-cell level

Simultaneously detect 12+ proteins in your sample—all at the single-cell level—using spectral and size-based multiplexing strategies. You can even strip & reprobe scWest chips up to 9 times for higher multiplexed studies!

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I need to simplify my phospho-flow signaling studies

Eliminate the fixation and permeabilization steps of flow/FACS! Milo chemically lyses the cells captured on the scWest chip before analysis to gain access to intracellular and intranuclear compartments more easily than with flow cytometry. By lysing the cells, Milo can detect challenging proteins like transcription factors and even methylated histones!

You can also use Milo's sizing step to separate out non-specific binding, which is more common with phospho-specific antibodies and cannot be resolved with flow.

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Milo chart - STAT1 signaling - Eliminate the fixation and permeabilization steps of flow/FACS

I need to detect proteins that lack good flow antibodies

Milo is an open platform so you can use the large commercial catalog of Western antibodies which is 10-100x larger than the flow/FACS catalog. Don't get stuck without a flow antibody against your target of interest again.

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Milo chart - Milo allows you to use the large commercial catalog of Western antibodies which is 10-100x larger than the flow catalog.

I want to measure protein isoform heterogeneity

Milo's molecular weight sizing step can resolve protein isoforms that differ in molecular weight, allowing you to measure how many cells in your sample express one isoform, the other isoform, or both isoforms. Try that with flow!

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Milo chart - Measure protein isoform heterogeneity - 2 isoforms resolved by molecular weight

I need to measure gene editing efficiency or effect in low efficiency gene editing systems

Milo can quantify the percentage of cells in your sample that express an inserted gene, even in low efficiency systems. Plus, you can simultaneously measure your edited gene and downstream effect markers to understand the impact of the gene on the cells that have been edited.

Milo chart - Measure gene editing efficiency or effect in low efficiency gene editing systems

I need to measure protein expression in low abundance samples

Milo can analyze samples which contain as low as 10,000 single-cells so you no longer have to have collect millions of cells to analyze protein expression. The number of cells captured scales with the number of cells loaded so even lower abundance samples are possible.

Milo can also characterize highly enriched FACS-sorted cell populations which don't contain enough cells to analyze with other techniques.

Milo picture - Measure protein expression in low abundance samples and characterize highly enriched FACS-sorted cell populations

Single-Cell Applications

Cancer

Application Notes

Application notes

		Wes and Milo Synergize to Profile Immune Cell Populations in the Tumor microenvironment- Application Note	[show details]
		In this application note, we’ll show you how Wes™ and Milo™ partner to get you critical answers to 1) what type of immune cell populations are present in a sample and then 2) what percentage of cells in that sample make up a specific immune cell subtype.
		Mending the Myocardium: Tracking Cardiomyocyte Differentiation with Single-Cell Westerns	[show details]
		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
		Wes and Milo Tumor Microenvironment App Note Japanese

		Wes and Milo Synergize to Profile Immune Cell Populations in the Tumor Microenvironment	[show details]
		Immunotherapy has opened the door to a new era in cancer treatment where dramatically successful and durable responses are being reported. Still, the population of patients who respond are a minority, and this highlights the need for a deeper understanding of the immune response to cancer. To that end, accumulating evidence suggests that the heterogeneous makeup of the tumor microenvironment (TME) is a major contributor to the discrepancies in response observed.
		Identify and Quantify Neural Subtypes with Single-Cell Westerns Application Note	[show details]
		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.
		Dissociation of Mouse Neural Tissue for Single-Cell Western Analysis Application Note	[show details]
		In this application note, we describe and characterize a protocol to successfully dissociate mouse neural tissue microsurgically dissected from combined cortex, ventricular zone, and hippocampus regions of E18 mice into single cells.
		Multiplexed Single-Cell Western Analysis of Red Blood Cells for Biomarker Detection in Blood and Neurodegenerative Disorders

		Going with The Flow: Using Milo to Streamline Your Flow Cytometry Experiments

		Generating Single-Cell Western Protein Heterogeneity Data with an InnoScan 710 Scanner	[show details]
		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.
		Using Single-Cell Westerns to Validate Single-Cell RNA-Seq Data	[show details]
		Single-cell gene expression studies are revolutionizing our understanding of heterogeneity in disease. Single-cell RNA sequencing tools are powerful for discovering mRNA transcript heterogeneity. However, mRNA levels do not always correlate with functional protein levels. In this application note, learn how Milo was used in parallel with a single-cell RNA-Seq workflow at the Stanford Functional Genomics Facility to validate single-cell RNA expression studies with single-cell protein expression data and ensure accurate and complete conclusions about cellular function. Because it uses the large Western catalog of antibodies & can easily measure intracellular proteins, Milo is the only platform with the versatility to detect diverse targets that are discovered in a sequencing run.
		Reveal Cell Subtypes, Protein Isoforms, and Phospho-Protein Heterogeneity with Milo	[show details]
		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.
		Adapting the Single-Cell Western Protocol to Detect Histone Modifications	[show details]
		The Single-Cell Western™ protocol is highly versatile and can easily be adapted for different biological applications and protein targets. Researchers studying signaling proteins or other targets that require treatment of cells prior to analysis can add a drug, cytokine, or other form of pre-treatment directly to scWest chips after cells have been captured and before running the chip on Milo. Cells can also be treated just prior to settling them onto scWest chips. As a result, the flexible Single-Cell Western workflow allows time-dependent experimental manipulations to be easily performed before lysis and electrophoresis. Here we describe a two-step, on-chip pre-treatment protocol to measure heterogeneity of modified histones — a class of proteins that is challenging to measure but critical in regulating gene expression. This new measurement capability could be key to unlocking new discoveries in the field of epigenetics.
		Protein Expression Heterogeneity with Milo, the First Single-Cell Western System	[show details]
		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.
		The Single-Cell Western has Arrived	[show details]
		Milo enables scientists to perform single-cell resolution Westerns (scWesterns) for over 1,000 individual cells simultaneously, and in a fraction of the time of conventional Westerns. Researchers can now gain selective protein expression information for up to four protein targets in each cell, offering views into cell-to-cell variation within a complex sample.

Specifications

Requirements & compatibility

Sample type	Suspension containing >10,000 cells
Cell diameter	7-25 µm in suspension
Cell type	Mammalian cells; globular in suspension and unfixed
Antibody requirement	Standard unlabeled primaries and fluorescent secondaries
Other equipment needed	Open-format fluorescence microarray scanner capable of 5 µm resolution

Performance & specifications

Typical cell dilutions	Yield capture and analysis of 1,000-2,000 cells per scWest chip
Molecular weight (MW) range	15-175 kDa
MW resolution	10% differences in distinct spectral channels, as low as 30% differences in same spectral channel
Typical target multiplexing	Up to four proteins per cell by spectral and size-based multiplexing Twelve-plus proteins per cell using stripping & reprobing
Workflow time	4-6 hours

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Scientific Content

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Peer Stories

Milo Antibody Database

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