Novel Molecular Assays

Near-infrared live cell senescence assay

Our recent work published in Nature Cell Death Discovery demonstrates the first known use of the cell permeant, near-infrared beta-galactosidase probe DDAOG to detect senescence in living cells.

Click here to view full paper.

Figure 1. Chemical structure of DDAO-Galactoside. Ex/em = 645/660 nm.

The fluorescent spectral characteristics of DDAOG make this probe particularly well suited for use in flow cytometry analysis using a red excitation laser and near-infrared emission detector, a standard configuration on many cytometer instruments.

 

Figure 2. Detection of gamma-radiation induced senescence in living melanoma tumor cells.

An increase in cellular senescence results from increased doses of gamma radiation, from < 2% senescence induced by 0 Gy to 90% senescence induced by a dose of 25 Gy. Near-infrared DDAOG detection of senescence-associated beta-galactosidase (SA-B-Gal) at 660 nm is shown on the X-axis. Cellular autofluorescence detected at 515 nm (green emission) is shown on the Y-axis. The observed increase in cellular autofluorescence seen in senescent cells is a result of accumulation of oxidized lipoprotein aggregates, and provides a reliable secondary senescence assay parameter.

 

Figure 3. DDAOG enables sensitive and specific detection of senescence in unfixed, living cells with very low background signal interference.

The cell-permeant DDAOG probe enables detection of SA-B-Gal in living cells, unlike conventional SA-B-Gal stain X-Gal (a, b) which must be applied to fixed cells to provide a colorimetric readout. The green fluorescent stain C12-FDG can be used to detect SA-B-Gal in living cells (e), but its emission overlaps green cellular autofluorescence (c), confounding specific detection of SA-B-Gal by this probe. In the near-infrared region of the fluorescent spectrum, cellular autofluorescence is negligible (d), enabling the signal provided by DDAOG (f) to be a reliable measure of SA-B-Gal in fluorescent senescence assays.

 

Figure 4. High content, high throughput flow cytometric screening of senescence and reactive oxygen species in living cells.

Here, a 96-well plate high-throughput flow cytometer (BD Fortessa HTS) was used to evaluate senescence (red, green) and reactive oxygen species (ROS, violet) in living cells treated +/- gamma radiation and +/- 36 unique redox modulating compounds. Our findings are presented in further detail in the publication referenced at the top of the page. While we used a violet ROS probe for this study, any violet/blue fluorescent probe with minimal emission at wavelengths > 500 nm is compatible with the red/green senescence assay approach. Our novel flow cytometry assay enables rapid detection of any violet/blue probe signal along with simultaneous quantitation of senescence in thousands of single, living cells.

- Data contributed by Amy Flor, Kron Lab. 2016.