Single-Cell Western 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 it’s 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) if 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).

How do we confirm that there is a single cell in each well?

Once the cell suspension is loaded on to scWest chip, users can visually inspect the chip under a standard bright-field cell culture microscope to see single-cells captured in each microwell. Single-Cell Westerns use a Poisson-like distribution to capture about ~1000 single-cells in one scWest chip. In general, approximately 15% of the microwells should contain 1 cell and < 2% should contain two cells. Most microwells should be empty. You can confirm this occupancy by inspecting a portion of the chip which takes < 5 min for one block of 400 microwells. The data obtained from this inspection may be extrapolated to the full chip to get a doublet rate for each sample.

In addition, during probing, each scWest chip is probed for an internal control (e.g., beta-tubulin or GAPDH) so that users can tell which microwells contained cells in the downstream analysis. Once the lysis step and the Milo run starts, it is difficult to differentiate between singlets or doublets in the wells using the analyzed peak area data owing to significant overlap in peak areas quantified in singlet and doublet lanes. The standard Single-Cell Western protocol does not exclude doublets from the single-cell data after running the sample on Milo.

For many applications, the observed doublet rate quantified in the inspection step may be acceptable and users of Single-Cell Westerns may just want to report accurate doublet rates for their experiments. In some situations, the error from the doublet rate will be on the same order as the statistical sampling error. For example, in a situation where a user is trying to enumerate cells that are positive for a certain marker in a mixed 50/50 population, the error from the doublet rate will be about 2.5% which is on the same order as the sampling error. If a user wants to reduce the doublet rate for their experiment, they should reduce cell settling time and/or cell concentration in the cell loading step.

How quantitative is Single-Cell Western technology?

Scout software analyzes Single-Cell Western data and can provide two types of quantitative information: enumeration of target positive cells and quantification of target abundance in each single cell. The ability to count how many cells in your population express a specific marker can help users identify and quantify the abundance of subpopulations of cells within a mixed sample. The ability to quantify how target expression varies across a population of cells can help users understand variability of cellular response to a stimulus or how target expression varies across a tissue. Shown below are a few examples of types of quantitative plots (e.g., histograms and scatterplots) that Scout can generate.

There are lots of different ways to visualize your data. For example, when quantifying heterogeneity in target expression, you can create scatter plots of the peak area across multiple samples or chip replicates. You can also create histograms of the peak area to see distributions of expression.

What is the sensitivity of the Milo system?
This number is difficult to pin down exactly because it is very difficult to create control samples that load a known amount of protein into each well in a cell-like vehicle. Moreover, as for other immunoassay platforms, the sensitivity of Milo is dependent on the antibody used. Our best estimate for Milo is that it has approximately femtogram-level sensitivity. We have detected what are considered to be low abundance targets, especially transcription factors and phosphorylated proteins (e.g., FoxP3, Pax8, pSMAD3 and pERK in unstimulated cells). According to our scientific team’s estimate, approximately 80% of the proteome is expressed in single-cells at a level that can be detected by Milo.

Can Milo work with fluorescent labeled primary antibodies?
The recommended Single-Cell Western protocol uses unlabeled primary antibodies followed by fluorescent secondary antibodies. Milo can work with fluorescent labeled primary antibodies, however there is some loss in sensitivity compared to using labeled secondary antibodies with an unlabeled primary. Using a labeled secondary antibody gives a several-fold increase in target signal mostly because multiple secondary antibodies can bind to a single primary antibody. Because of this sensitivity gain, Single-Cell Western users commonly choose labeled secondary antibodies for most immunoassays. Milo’s concept is similar to immunostaining on microscope slides. Using an antibody probing chamber that comes with the platform, we allow the antibody to diffuse into the gel. First apply an unlabeled primary antibody cocktail, then wash, and then a fluorescent secondary antibody cocktail. Then wash and image the chip’s fluorescence. To increase throughput of the antibody probing process, Single-Cell Western users can get more antibody probing chambers (PN A200) and probe many chips in parallel. To screen more antibodies in parallel, users can purchase a 3-plex antibody probing fixture (P/N A300) which contains three distinct antibody chambers and enables three different antibody cocktails to be probed on one chip.

Can Milo tolerate if the size of the cell is either above or below the recommended range of cell sizes (7-25μm)?

It may be possible to measure cells that are below the recommended range of 7-25 μm using small scWest Chips (Product Number K500). Because Milo uses a Poisson-like distribution to capture the single-cells rather than relying on cell size for cell capture, smaller cells should still settle into microwells. The limitation for small cells is system sensitivity and having enough protein expressed, especially in very small cells. We have successfully worked with very small cells including primary bone marrow cells, red blood cells and lymphocytes to date. Cells that are much larger than the indicated range may not fit into the large diameter microwells (Product Number K700) and cannot be analyzed on the current system. We have successfully worked with primary cardiomyocytes using Large scWest chips. The table below shows the cell diameters that are recommended for each chip size. We also offer a calibration chip which contains all three well sizes on one chip so you can test cell settling and choose the optimal well size for an unknown sample. The largest cell size we can analyze is 25 μm.

Can you do phospho- and total probing at the same time in the same cell?
Yes. Milo can be used to probe for a phospho-specific epitope and total target to quantify the extent of phosphorylation as a percentage of total target abundance (e.g., pERK and total ERK). To improve signal, it may sometimes be beneficial to sequentially probe for phospho targets first and then probe for the total target rather than probing with both antibodies simultaneously.

What is the protocol to stimulate my cells “on chip” after they have been captured in the microwells?

For on-chip stimulation, there are two protocol options:

• During Cell Settling: Cells may be stimulated during the settling step by spiking the stimulant into the Suspension Buffer. For example, you can add EGF to the Suspension Buffer so that the cells are being stimulated during settling. You would then wash the unsettled cells off the gel surface as normal, put the chip into Milo, add your combination Lysis/Run Buffer and run the assay as normal. This protocol was followed in the figure below to upregulate pEGFR after a 5 min EGF stimulation of A431 cells.

Expression of pEGFR after 5 minutes of EGF stimulation of A431 cells analyzed on Milo with normalization using GAPDH expression

• After Cell Settling: Cells may also be stimulated after they are settled/captured in the microwells, and immediately before lysing and initiating the run. In this protocol, you would settle your cells as normal in Suspension Buffer (with or without stimulant), wash off uncaptured cells and then put the chip into Milo. You would then pipette 1 mL of stimulation buffer on top of the cells (the same way that you load the sample on the chip). This covers the chip surface with stimulation buffer. You can let the cells stimulate for up to 30 min. Since you already have 1 mL of stimulation buffer on the chip surface, remove 1 mL of Lysis/Run Buffer from the single-use Lysis/Run Buffer vial before pouring it into the electrophoresis cell (the goal is to use the same total volume as what is provided). Then pour the 14 mL of Lysis/Run Buffer (1 mL less than the 15mL provided in the single use Lysis/Run Buffer vial) into the electrophoresis cell as normal which dilutes the stimulation buffer and lyses the cells, and run the assay as normal.
  
  Note: Cell survival when left in the suspension buffer on the chip varies from cell line to cell line, and has to be optimized depending upon application. Cells will slowly start to die off if left too long. We recommend not leaving your cells for longer than 30 min on the chip in the suspension buffer.

What are the properties of the Lysis/ Run buffer?
scWest kits come with 8 vials of single use Lysis/Run buffer to run 8 scWest chips. The Lysis/Run buffer contains SDS. Our Lysis/Run buffer does not contain any phosphatase or protease inhibitors but Single-Cell Western users can spike them in before running the assay. We have not seen a major improvement in separation resolution or peak signals with the addition of phosphatase or protease inhibitors, potentially because the lysis time is so fast. Lysis/Run buffer does not contain a reducing agent. You may add a reducing agent to the Lysis/Run buffer immediately before the Milo run. However, adding a reducing agent may change the conductivity, so you will need to reduce the electrophoresis voltage during the run.

What is Milo’s multiplexing ability?
Milo can multiplex up to 12 proteins per single-cell using one or more of the following multiplexing strategies:

• Size-based multiplexing: Using size-based multiplexing, the SDS-PAGE separation is used to resolve proteins that differ in their molecular weight. Size-based multiplexing is possible because Milo can resolve proteins that differ by approximately 30% in molecular weight, allowing you to image targets that differ in molecular weight in the same spectral channel. For example, if you are trying to measure ERK (42 kDa) and EGFR (175 kDa), Milo will resolve these two proteins by molecular weight in the SDS-PAGE separation so you can detect these two targets in the same spectral channel.
• Spectral multiplexing: Antibodies conjugated with distinct fluorophores are used in spectral multiplexing and images are captured using different spectral channels using a multi-channel microarray scanner. With spectral multiplexing, the targets are simultaneously probed with primary antibodies raised in different host species and then secondary antibodies raised against each host species and tagged with distinct fluorophores are applied to visualize the bands. For example, you might probe for ERK and EGFR with mouse ERK and rabbit EGFR primary antibodies and then come in with donkey anti-mouse 555 and donkey anti-rabbit 647 secondary antibodies and scan the chip in the 555 and 647 channels on your 2 color scanner.
• Stripping & re-probing: After probing an scWest chip, antibodies can be stripped off with minimal sample losses because the sample is covalently bound into the gel after the SDS-PAGE separation and UV light exposure. This unique feature enables multiple rounds of stripping & re-probing, something which is typically not possible with conventional Western blotting which relies on weaker electrostatic or hydrophobic interactions to immobilize the sample on a membrane.

Can you strip & re-probe a chip?
As described above, stripping & re-probing scWest chips is possible. Hughes et al (Nature Methods) shows 9 cycles of stripping and re-probing on the same chip.

Since your sample is covalently bound into the gel after the SDS-PAGE separation and UV light exposure, antibodies can be stripped off with minimal sample losses. This enables multiple rounds of stripping & re-probing on Milo – something that isn’t typically possible with conventional Westerns which rely on weaker electrostatic or hydrophobic interactions to immobilize the sample on a membrane. Please refer to our tech note on stripping & reprobing for more details.

How do you do molecular weight sizing with Milo?
Milo can do both quantitative molecular weight sizing and relative molecular weight sizing. The vast majority of Single-Cell Western users’ applications only require relative sizing for their targets of interest. To get relative sizing, every chip is probed for an internal control protein like beta tubulin and then the mobility of the target of interest is compared to the internal control. For example: a 20 kDa protein should migrate faster than beta tubulin (50 kDa) while a 100 kDa protein should migrate slower. Milo can also do quantitative (absolute) molecular weight sizing. For absolute sizing (i.e., quantifying whether your protein is migrating as 50 kDa or 70 kDa), Single-Cell Western users will need two or more reference points to create a molecular weight sizing ladder. This ladder can be obtained by probing for two endogenous internal control proteins that differ in molecular weight (e.g., beta tubulin and GAPDH). Alternatively, Single-Cell Western users can probe for an endogenous protein (e.g., beta tubulin) and also spike in one or more purified proteins into the settling buffer, loaded simultaneously with the cell sample. Molecular weight ladder proteins can then be identified and labeled with their molecular weight in Scout and Scout will then use this sizing ladder to quantify target molecular weight. Watch the Scout Training Video for more information.

I do flow sorting. How can I use Milo? Is Milo compatible with flow sorting or sorted cell populations?
Milo is compatible with flow-sorted cell samples. You can use flow sorting to enrich for a certain subpopulation of cells. For example, Single-Cell Western users can sort into a tube and then pipette that enriched population of cells onto the scWest chip for further characterization of intracellular proteins. We find that potential interference of the fluorescent antibody/antibodies used for sorting is target and protocol dependent. When testing a new sorting protocol and sorted sample with Milo, we recommend that Single-Cell Western users use fluorescent antibodies for their flow sort that can be visualized in their microarray scanner so they can see if the antibodies from the flow sort are interfering with the downstream Single-Cell Western readout.

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.

Can exosomes or other extracellular vesicles (EVs) be run on Milo?
Milo has been used successfully to measure protein expression in exosomes, a type of EV. Milo cannot analyze individual EVs (they are ~100 nanometers in diameter, whereas we spec the Milo instrument to be able to accommodate cells with diameters of 7-25microns), but Single-Cell Western users can load EVs onto the scWest chips and let them settle into the microwells to measure many EVs per microwell for a high sensitivity measurement of pooled EVs.

What if I’m seeing non-specific antibody binding of either my primary or secondary antibodies?
Some antibodies and cell types perform better with other types of blocking conditions. Rather than using the blocking buffer provided in the scWest kit, users can substitute it with other traditional Western blocking buffers like Superblock (Life Technologies), casein, etc. You can also reduce the primary and secondary antibody concentrations used. For new or non-validated antibodies, we suggest starting with a high concentration of ~50-100 μg/mL. If needed, antibody concentrations can then be reduced in subsequent runs.

How many cells must be loaded to get 1,000 cells captured for analysis?
To analyze 1000 cells, we recommend loading 100,000 cells in 1 mL on a single scWest chip. Using the standard Single-Cell Western protocol, you typically capture 1-4% of the cell population that you load on the chip. With 100,000 loaded cells, you typically capture 1,000 - 2,000 single cells. With 10,000 loaded cells, you typically capture 100-400 cells. Increasing the settling time has the biggest impact on the number of captured cells for a given concentration. The microwell occupancy is just the number of captured cells divided by 6400 wells. So, if you capture 1000 cells, the well occupancy is around 15%. This is ideal because at 15% occupancy, the Poisson distribution predicts the maximum number of singlets and minimum number of doublets.

Note: There are 6,400 microwells on each chip. You will typically capture 1-4% of loaded cells so you should expect to capture approximately 10-40 cells from a small sample of 1,000 starting cells.

How would you combine FACS with Single-Cell Western analysis?
A typical workflow integrating flow cytometry and Single-Cell Western analysis is to run your cells through a FACs sorting experiment to enrich for a subpopulation of cells based on a set of cell surface markers and then collect this enriched population of cells and load it onto Milo to do further characterization of challenging intracellular proteins like phospho-proteins or transcription factors. For example, one Milo user sorted dissociated human breast tissue for EpCAM+ cells and then used Milo to analyze cytokeratin heterogeneity within the EpCAM+ cell population (Nguyen, et al Nature Communications 2018). Interestingly, she was able to measure a trimodal population of cytokeratin expressing cells within the enriched EpCAM+ cell population. She did not have good flow antibodies for her cytokeratin assay. Alternatively, in this example, she could simultaneously probe the scWest chip for both EpCAM and cytokeratin and eliminate the need to do a flow sort before the Single-Cell Western run. This study design would allow her to identify the EpCAM+ cells and then measure cytokeratin expression within this subpopulation of cells.

What do you mean by molecular weight resolution in distinct or separate spectral channels?
The scWest chips are imaged using a fluorescence microarray scanner. Protein targets that differ by >30% in molecular weight can be resolved by size and the separated proteins can then be probed with primary antibodies followed by secondary antibodies tagged with the same fluorescent dye (e.g., Cy3). In this way, you will be able to detect two protein targets in the same spectral channel. If protein targets are more similar in molecular weight, you should probe them with primary antibodies raised in distinct host species (e.g., rabbit and mouse) so that you can then probe with two secondary antibodies that are tagged with distinct fluorescent dyes (e.g., Cy3 & Cy5). In this way, you can detect your protein targets using distinct spectral channels.

How can we have confidence in heterogeneity data? How can we separate out technical noise from biological variation? If the general background signal varies across a chip, the detected peaks can also vary in brightness with the peaks at the lower end being darker according to the observed bias. How can we trust the data?
In order to be confident that the measured variability in the peak areas is significant and not attributable to technical noise alone (i.e., variation in antibody probing), you can compare the difference in background fluorescence across the chip to the variation in the overall peak areas that are measured. In general, we observe that variation due to background brightness is very small compared to the variation in the peaks so the background error is insignificant. When following the recommended protocol during our validation studies, we found that the spatial variation of the mean peak area across the chip (i.e., an x-y surface fit of the peak area) was relatively small compared to the total variation of the peak area. In one data set of 67 chips, the spatial variation in peak area only accounted for on average 16% of the range of the peak area distribution (which is usually several orders of magnitude). Qualitatively you can see this by observing that a very bright peak will often occur right next to a very weak peak.

What is the assay development process for Single-Cell Westerns like?
For new assay development, we recommend testing different antibodies to identify the best performing antibodies for your target(s) of interest. About 2/3 of all Western validated antibodies work on the platform so we recommend trying 2-3 antibodies when setting up an assay with a new target and then selecting the highest performing antibody. You can use the Three Plex Probing Chamber (Product Number A300) to screen 3 antibody cocktails in different regions of one chip to streamline assay development times.

Rarely do you need to adjust the antibody concentrations. If the antibody works by itself, it will work when multiplexed with other antibodies most of the time. In rare cases where simultaneous probing causes a reduction in signal for one of the targets, you can probe targets in series to achieve the desired multiplexed assay (e.g., probe for phospho target first and then re-probe the same chip with total protein antibody).

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.

Do you have any antibodies which are guaranteed to work with Milo?
Milo is a novel technology and our labs at Novus Biologicals and R&D Systems are validating a long list of antibodies on this new platform. At the moment, these are hundreds of targets for which we have Single-Cell Western grade antibodies including: ATM, Beta Tubulin, CD11b, CD81, GAPDH, Histone H3, IRF3, MAP2, mCherry, MTH1, MyoD, Myosin Light Chain 2, Neurogenin 2, OLFM4, pan-Cytokeratin, PAX8, PHF6, SLC22A2/OCT2, SMAD3, Tyrosine Hydroxylase, UQCRFS1, and ZEB1. View our most updated list of Novus brand Single Cell Western certified antibodies or our comprehensive list here.