Single-Cell Western Resources
Tools | Protocols | Application Notes | How-to-Videos | FAQs
Step into our virtual lab and experience Milo and its Single-Cell Western technology
How to set up a Milo Experiment
Learn How Milo Validates Single-Cell RNA-Seq Data
Validating Single-Cell RNA-Seq Data with Milo
FAQs
How big is the Milo instrument? What other equipment is needed when using Milo?
Milo is a benchtop instrument, approximately the same size of a laptop, with physical dimensions of 33.2 cm length, 27.6 cm width, and 15.2 cm height (weighs 10 lbs). Milo users must have access to an open format microarray scanner capable of 5-10 μm resolution fluorescence scanning.
Milo can be purchased together with a high sensitivity microarray scanner or you can use a compatible scanner already available at your institution. Users can choose ProteinSimple’s 2-color InnoScan 710 or 3-color InnoScan 1100 scanners which offer high resolution scanning, sequential or simultaneous scanning abilities, and high sensitivity PMT systems which make them the most sensitive scanners on the market. Both scanners come with a Dell PC and a monitor which runs MAPIX software to operate the scanner and sufficient capabilities to run Scout software for Single-Cell Western data analysis.
If you have access to a microarray scanner at your institution, you may view the list of Milo compatible microarray scanners here. The microarray scanner does not need to be in your lab as chips can be run, probed & dried and then batches of chips can be transported to the microarray scanner for scanning. The required specifications for a microarray scanner are:
- Number of colors: Most scanners are 2, 3 or 4 color scanners. The more colors you can detect, the higher multiplexing (targets per cell) you can do.
- Resolution: At least 10 μm scanning resolution is required, but 5 μm scanning resolution is recommended.
- Detection method: Fluorescence based
How does Milo identify which type of cell its probing?
To identify specific cell types within a mixed population, Milo users can probe the single cells captured in the scWest array for a protein which identifies a specific cell type (i.e. using a cell marker antibody). A common study design ("if/then" study design) is to probe for 2 targets in each cell and analyze (i) whether a cell is positive for Target A (e.g., a cell-type marker), then (ii) is it positive for Target B (e.g., a downstream signaling protein indicating cell signaling activation or a putative drug target).
What sample types work best for Single-Cell Westerns using Milo?
Unfixed samples that are in single-cell suspension can be loaded onto scWest chips. The type of samples which can be used for Single-Cell Westerns using Milo include cell suspensions, trypsinized adherent cell lines, frozen cells, cells isolated from tissues, and dissociated tissues. If working with frozen cells, first thaw them and then re-suspend in Suspension Buffer and load the suspension onto an scWest chip. If there are significant dead cells after the thaw, Single-Cell Western users can do a live/dead sort on a flow cytometer before running the sample on Milo. Tissues can also be analyzed after they have been dissociated into single cell suspension. The wells on the scWest chip are approximately 30μm deep so that the cells sit deep in the wells. The cells settle into the microwells by gravity and they are pretty-difficult to wash out after they settle in. We designed the microwells to be deep enough that the cells fall into the bottom of the well and don’t wash out easily.
What are the advantages of Milo when compared to confocal microscopy?
- More antibodies available: Antibodies which work for Western blot can be used on Milo. There are more Western blot validated antibodies available in the market compared to confocal microscopy or immunocytochemistry/immunofluorescence (ICC/IF) validated antibodies.
- High specificity: Single-Cell Westerns allow you to integrate the signal from the band of interest and exclude off target bands or background lysate binding. This is not feasible for immunostaining assays.
- Higher cell throughput: Milo can analyze ~1,000 single cells in parallel. With ICC/IF, users manually image the data and analyze each single-cell which limits the analysis to no more than 100 single cells.
- Quantitation: Milo can more easily quantify target expression in each cell because he can integrate area under the curve for each band in each single-cell.