Solve Flow Cytometry and FACS Cell Sorting Challenges with Milo
Whether you’re already running flow cytometry assays or considering getting started, we want to help you get the data you need quickly by eliminating common workflow challenges.
Table of Contents
What is Fluorescence Activated Cell Sorting (FACS)?
Fluorescence Activated Cell Sorting (FACS) is a method of separating cells into subpopulations which utilizes fluorescently-labeled antibodies that detect certain protein markers in individual cells. A FACS machine is sometimes called a cell sorter.
What is Flow Cytometry (Flow Analysis or Flow)?
Flow cytometry is a method of measuring protein expression in individual cells that uses fluorescently-labeled target-specific antibodies to measure the heterogeneity in protein expression in a cell sample. A flow cytometry machine is sometimes called a FACS analyzer and is used for cell analysis.
What is the Difference (FACS vs. Flow Cytometry)?
FACS is used to sort cells and enrich for a subset of cells which is often then studied in further detail using flow cytometry or other analytical techniques. Flow cytometry is used for cell analysis and is focused on measuring protein expression or co-expression within a mixed population of cells. Benchtop flow cytometers are smaller format machines that are suited for use in individual labs.
What are some Common Challenges with Flow & FACS?
- I need to make highly specific, highly multiplexed measurements but can’t fully eliminate antibody cross-talk and fluorescence bleed-through
- There isn’t a good flow-validated or FACS-validated antibody against my target of interest
- I have an intracellular protein that I’m trying to detect but I can’t seem to get the assay working
- I don’t want to have to fix & permeabilize my cells to measure intracellular protein expression
- I have too few cells in my sample to put in a flow cytometer or FACS instrument.
Single-Cell Westerns on Milo
Single-Cell Westerns are a method of measuring single-cell protein expression that separates the proteins in single-cell lysates by molecular weight and then uses western-validated antibodies to make highly specific measurements of the heterogeneity in protein expression in a cell sample. Single-Cell Westerns can integrate downstream of FACS to analyze sorted cell samples and also simplify challenging flow cytometry assays.
The Benefits of Single-Cell Westerns
- Simplify challenging flow cytometry assays (e.g., transcription factors or methylated histones) by eliminating fixation & permeabilization steps. Single-Cell Westerns analyze single-cell lysates so any proteins in or on the cells are detectable with the same workflow.
Additional Benefits of Single-Cell Westerns
- Use conventional Western antibodies to detect proteins that don’t have good flow or mass cytometry antibodies.
- Analyze samples with low cell numbers (e.g., highly enriched FACS sorted cell populations, rare stem cells, or other primary samples with as low as 10,000 starting cells).
- Improve specificity of target detection by only integrating signal from the protein band at the correct molecular weight.
- Measure protein isoform heterogeneity by resolving protein isoforms by size and integrating signal from one or the other isoform.
- Multiplex 12+ proteins per single-cell analysis using differences in target molecular weights and stripping & re-probing rather than spectral multiplexing alone.
Milo Solves Flow Cytometry Challenges
With Milo, you can eliminate the fixation and permeabilization steps required by standard flow/FACS protocols, while efficiently gaining access to intracellular and intranuclear compartments. By lysing your cell sample instead, Milo can better detect challenging proteins like transcription factors using widely available Western blot antibodies. Further, Milo provides molecular weight information in addition to antibody binding information, improving the specificity of your measurement.
We demonstrated the application of Milo as an alternative to flow for the sensitive and specific analysis of single cell protein expression using the transcription factor PU.1 and various cell lines and primary cells as a proof-of-concept. Milo’s ability to discern subpopulations within the same sample type based on thresholding signal abundance and his ability to enumerate distinct populations of cells based on the percentage of target-positive cells makes him an attractive option for the characterization of proteins that are painful to access using traditional flow methods.