What is manual gating?

Manual gating is the process of identifying immune cell populations based on the protein markers they are known to express, based on existing literature. For example, we know that T cells express CD3 and B cells express CD19 so we can plot the cells using these two markers as our axes to identify these two populations. (See Fig A)

We can then drill down into each population to identify subsets. For example, we can go into the T cell gate and look at another set of two markers like CD4 and CD8. We continue to do this going down into CD4 or CD8 populations until we’ve identified all of the different T cell subsets. (See Fig B)

Then we can go back to our B cells in the CD19+ gate and use other markers to drill down to B cell subsets. Then into the third gate and use other markers to identify populations like NK cells, Dendritic Cells, and Monocytes by looking at expression of other protein markers unique to those cell types (e.g. CD56 for NK cells, CD14 and CD16 for monocytes, etc.). This requires extensive knowledge of marker expression across all cell types to ensure you’re correctly identifying each population.