When do I want to classify cells? Why do I care where a protein localizes? These are actually two different questions that utilize different applications to answer.
Cell classification uses specific markers to identify a specific cell type in a heterogeneous population. If, for example, you want to understand the mechanisms microglial cells use to remove damaged neurons, you’ll want to make sure your sample is made up of just microglial cells using markers like Iba1, CD11b, and TMEM199. Otherwise, you risk biasing your data if your sample contains other types of brain cells. Similarly, if you’re using stem cells to explore differentiation pathways, you’ll want to periodically check your sample to make sure it hasn’t differentiated unexpectedly by looking for expression of stem cell markers like Sox, Oct4, and Nanog. Classifying cells ensures you aren’t performing experiments with misidentified or cross-contaminated cells.
How do you classify cells in a mixed sample? Flow cytometry is well suited for this application because it delivers data at the single cell level. Cells can be classified using markers that reside intracellularly or at the cell surface. They can be used to stain the cells independently, or in a multiplexed panel; however, you will have to understand the antibody conjugates compatibility with different sample preparation methods. To multiplex, just use primary antibodies, conjugated to different fluorophores, specific to the cell marker of interest. Fluorescence activated cell sorting (FACs) can then be used on stained live cells to sort cell populations based on marker profiles in order to isolate a purified, homogenous population of cells for further experimentation. Check out our Human Immune Cell Marker Guide, Mouse Immune Cell Marker Guide, Neuronal and Glial Cell Markers Pathway, or Stem Cell and Lineage Marker Guide and start classifying your cells!
Protein localization, on the other hand, tells you where a protein is localized in a cell or tissue at a given time. For proteins like the STAT family, which translocate to the nucleus when activated, knowing where a protein is localized can tell you if a protein is active or inactive under certain conditions. Immunofluorescence (IF) and immunohistochemistry (IHC) are the most commonly used applications for these types of experiments since you can visualize exactly which subcellular compartment your protein is localized in. Compartment-specific antibodies or dyes can be used in conjunction with an antibody against your specific protein of interest, allowing you to know exactly where in the cell your protein resides at a given time.
What if you need to classify your cells before determining protein localization because your protein of interest can be found in different subcellular compartments based on cell type? If you find yourself in this situation, one approach is to treat your cells, use the appropriate markers to collect a specific cell type using flow cytometry/FACs, and then follow that up with your IHC/IF experiment to understand where the protein of interest localizes in the cell population of interest.