Fluorescent Detection of Cell Senescence For Flow Cytometry and Imaging Applications

Beta Galactosidase Assay - Senescence Assay Using Fluorescence Substrate

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Cellular senescence is a state of stable growth arrest, and is thought to play a physiological role in normal development and tissue homeostasis. It is characterized by morphological and metabolic alterations, altered gene expression, and upregulation of pro-inflammatory secretomes and plays a physiological role in normal development and tissue homeostasis. Several factors drive senescence in cells, including telomere damage in senescence caused by aging and metabolic dysfunction. Since many pathways are associated with this process, senescence can be detected by looking into genotypic changes. Genotypic changes are sometimes cell-specific, and the intensity of the activating response varies. Evidence suggests that senescence-associated β-gal activity is highly upregulated and can be used to detect senescent cells. This assay is particularly useful for screening novel drug candidates that initiate senescence in cells or conditions that cause genotypic changes leading to senescence. In this AssayWise letter, we will discuss data from our cell senescence probe Xite™ β-D-galactopyranoside and its detection of senescent cells.

The identification of senescent cells is based on an increased level of lysosomal β-galactosidase (SA-β gal) activity. The first method to detect senescence-associated SA-β gal activity includes a cytochemical protocol suitable for the histochemical detection of individual senescent cells in culture and tissue biopsies. Cells under normal growth conditions produce SA-β gal, located in lysosomes. This enzymatic activity can be detected at pH 4.0 using the chromogenic substrate 5-bromo 4 -chloro3-indolyl β-D-galactosidase (X-gal). The SA-β gal positive cells can be scored under bright-field microscopy. Cytochemical methods can take approximately 30 to 60 minutes to execute and several hours to a day to develop and record the response. The disadvantage of using this method is the lack of sensitivity and quantitation.

The second method is based on the use of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), a fluorogenic substrate for β-galactosidase. This compound is membrane-permeable and nonfluorescent before hydrolysis; after hydrolysis of the galactosyl residues by β-galactosidase, the compound emits green fluorescence upon excitation and remains confined within the cell. The C12-FDG provides better sensitivity than the cytochemical method, but it is still not up to standard. We recently released a new probe that addresses the low sensitivity of the C12-FDG probe for fluorescence analysis and is very easy to use. Xite™ β-D-galactopyranoside detects the SA-β gal at almost ten times lower concentrations with better cell permeability than C12-FDG. Xite™ β-D-galactopyranoside contains a fluorophore attached to galactosyl moieties and lacks fluorescence. However, in the presence of a β-gal enzyme, the galactosyl group gets cleaved, separating the galactosyl residue from the fluorophore and thus providing fluorescence. This exact mechanism of the probe and lack of fluorescence in the absence of β-gal enzyme provides better specificity towards β-gal enzyme in the cells.

Materials and Methods

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Cell culture and reagents

Jurkat cells were maintained in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS). HeLa cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS. Reaction Buffer was used from Cat No. 23005, AAT Bioquest, Inc. Camptothecin and β-Galactosidase enzyme were purchased from Sigma and used as per manufacturer's recommendations.

Ex/Em measurement in the presence and absence of enzyme

In 2 mL of PBS, 100 uL of Xite™ β-D-galactopyranoside stock solution (2 µM) was added in reaction buffer without and with β-gal enzyme (2 U/mL) and Ex/Em was measured using Varian Eclipse^® fluorescence spectrophotometer.

Flow cytometry analysis

To induce senescence, Jurkat cells were treated with Camptothecin at 5 µM final concentration and incubated at 37C incubator for 6 hours. Cells were washed with DPBS and then stained with 5 µM probe constituted in reaction buffer for 30 minutes at 37C incubator. Cells were then washed twice with DPBS and resuspended in Reaction Buffer. Flow cytometry was performed with FITC channel using NovoCyte^® flow cytometer from ACEA Biosciences.

Acquisition of fluorescence images

HeLa cells were treated in a black 96-well plate and stained as indicated earlier. Fluorescence images were acquired using FITC filter set on fluorescence microscope.

Fluorescence plate reader analysis

Different doses of β-galactosidase enzyme was incubated alongside FDG or Xite™ β-D-galactopyranoside at the same concentration in a black wall clear bottom 96-well plate and fluorescence was measured using a Spectramax GeminiXS^® (Molecular Devices) fluorescence plate reader at Ex/Em= 490/525 nm with the cutoff 515 nm.

Results

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Ex/Em in the presence and absence of target enzyme

In order to verify the specificity of our Xite™ β-D-galactopyranoside, we measured excitation and emission in the presence and absence of β-gal enzyme. As we hypothesized that the Xite™ β-D-galactopyranoside only emits fluorescence in the presence of its respective enzyme, a significant amount of fluorescence was only observed upon presence of β-gal enzyme. This further strengthens the selectivity towards research studies that are conducted to differentiate senescent cells.

Detection of drug-induced senescence inside cells using flow cytometry and fluorescence imaging

Next, we shifted our testing to cells expressing higher concentrations of SA-β-gal enzyme with drug-induced senescence. We treated Jurkat cells with Campothecin to induce senescence, followed by incubation with either our Xite™ β-D-galactopyranoside or FDG. Samples were processed through flow cytometry (Figure. 2A) or fluorescence microscopy (Figure. 2B). The flow cytometry analysis showed that the Xite™ β-D-galactopyranoside gave almost 10-fold higher fluorescence intensity in camptothecin-treated cells compared to the untreated samples. Unlike our Xite™ β-D-galactopyranoside, the FDG did not produce any significant fluorescence with treated samples, mainly due to lack of cell permeability. This comparison sheds more light on how effectively our Xite™ β-D-galactopyranoside could be used for drug analysis in cell senescence studies.

Response comparison between FDG and Xite™ β-D-galactopyranoside using fluorescence microplate reader

Earlier we speculated that the response with FDG was lower because of the lack of cell permeability. To explore this possibility, we investigated the sensitivity of our Xite™ β-D-galactopyranoside for the detection of β-gal enzyme and compared the results with FDG. We ran the assay with different concentrations of β-gal enzyme and treated with 1 µM of either our Xite™ β-D-galactopyranoside or FDG and recorded the response using the fluorescence plate reader. A 10-fold higher response was obsereved with Xite™ β-D-galactopyranoside compared to FDG, which cements our notion that Xite™ β-D-galactopyranoside has a higher efficiency and signal intensity over other commercially available probes.

References

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1. McHugh, D., & Gil, J. (2018). Senescence and aging: Causes, consequences, and therapeutic avenues. The Journal of cell biology, 217(1), 65"77. doi:10.1083/jcb.201708092
2. Debacq-Chainiaux, J. D. Erusalimsky, J. Campisi, O. Toussaint, Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo, Nat. Protoc. 2009, 4, 1798.
3. Lòpez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194"1217.
4. Itahana K, Campisi J, Dimri GP. (2007). Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay. Methods Mol Biol 371: 21"31.

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