分子生物学

Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure (e.g., fluorescence correlation spectroscopy (FCS)-calibrated imaging and quantitative mass spectrometry). Here we detail a straightforward flow cytometry-based method to measure the absolute abundance of any Halo-tagged protein in live cells that uses a standard mammalian cell line with a known number of Halo-CTCF proteins recently characterized in our lab. The protocol only comprises a few steps. First, a cell line expressing the Halo-tagged protein of interest is grown and labeled side-by-side with our standard line. Then, average fluorescence intensities are measured by conventional flow cytometry analysis and finally a simple calculation is applied to estimate the absolute number of the Halo-tagged protein of interest per cell. Once the protein of interest has been endogenously tagged with HaloTag, which we routinely achieve by Cas9-mediated genome editing, the presented protocol is fast, convenient, reproducible, cost-effective and readily accessible.

The ability to non-invasively detect specific damage to the kidney has been limited. Identification of extracellular vesicles released by cells, especially when under duress, might allow for monitoring and identification of specific cell types within the kidney that are stressed. We have adapted a previously published traditional flow cytometry method for use with an imaging flow cytometer (Amnis FlowSight) for identifying EV released by specific cell types and excreted into the urine or blood using markers characteristic of particular cells in the kidney. Here we present a protocol utilizing the Amnis FlowSight Imaging Flow Cytometer to identify and quantify EV from the urine of patients with essential hypertension and renovascular disease. Notably, EV isolated from cell culture media and plasma can also be analyzed similarly.