Extraction of lipids from fixed cells. A) Schematic of experimental process to obtain extracted lipids from fixed cells. i: Live cells; ii: incubate with SF; iii: SF-loaded cells; iv: add fixative; v: fixed cells; vi: CE-LIF analysis of single, fixed, SF-loaded cells; vii: add extraction solution; viii: CE-LIF analysis of fluorescent lipid in lysate supernatant. B) Total amount of fluorescent lipid recovered from K-562 cells fixed with glyoxal, washed and then incubated with the indicated extraction solution. *P ≥ 0.05. Bar height represents the average of triplicate measurements and the error bars indicate one standard deviation.
Fix and Assay
Chemical cytometry using capillary electrophoresis, CE, is a powerful tool for measuring single-cell enzyme activity. However, these measurements are often confounding as dynamic processes within cells rapidly change depending on environment, meaning that cell handling, transport, and storage can affect signaling pathways and alter results.
To meet these challenges, Research Associate Angela Proctor and professor Nancy Allbritton of the Allbritton Group, published in the journal Analyst, describe a method utilizing aldehyde fixation to simultaneously terminate cellular reactions across a population, freezing reaction results in time prior to analytical analysis. Fluorescent sphingosine was loaded into cells of different lineages, leukemia and lymphoma cell lines and primary leukemia cells, and allowed to react before fixing. The remaining sphingosine and any products formed were then quantified with chemical cytometry utilizing CE.
Dr. Angie Proctor
When cells were loaded with sphingosine followed by glyoxal fixation and immediate analysis, 55 ± 5% of lipid was recoverable compared to an unfixed control. Storage of fixed cells for 24 h showed no statistical differences in total amount of recoverable sphingolipid compared to samples analyzed immediately after fixation—though there was a difference in recovery of low-abundance products. Sphingosine kinase activity decreased in response to inhibitor treatment compared to treatment with a DMSO vehicle, 21 ± 3% product formed in inhibitor-treated cells vs. 57 ± 2% in control cells, which was mirrored in single-cell measurements.
This “fix and assay” strategy enables measurement of sphingosine kinase activity in single cells followed by subsequent analytical assay separated in space and time from reaction initiation, enabling greater temporal control over intracellular reactions and improving future compatibility with clinical workflow.