Senescence Detection Using Reflected Light in Adipose Stromal Vascular Fraction

We study adipocyte senescence across different fat depos and metabolic conditions, like obesity and hyperinsulinemia. Traditional Brightfield-based SABG assays don't really allow for multiplexing and are very subjective. Our new protocol is less subjective and does allow for multiplexing.

To begin, retrieve the tube containing the human adipose tissue. Homogenize 10 grams of adipose tissue and add it to a tube containing 0.05%type one collagenous enzyme to obtain approximately one million stromal vascular fraction, or SVF, cells. Aliquot 500, 000 cells into each 1.5 milliliter microcentrifuge tube.

Add one milliliter of 2%paraformaldehyde to the tube for cell fixation. Then, incubate on a rocker at room temperature for 20 minutes. Centrifuge at 600G for 10 minutes and remove the supernatant.

Prepare X-Gal staining solution using the senescence beta-galactosidase staining kit according to the manufacturer's instructions. Add one milliliter of the staining solution into three fresh tubes. And adjust the pH to five, six, and eight using one molar hydrochloric acid or one molar sodium hydroxide solutions.

Use a calibrated pH meter to verify the pH. Add the pH-adjusted X-Gal staining solution to each of the labeled tubes containing 500, 000 cells. Incubate at 37 degrees Celsius for 24 hours without carbon dioxide.

Centrifuge the cells at 600G for 10 minutes. After removing the supernatant, wash the cells with PBS. And centrifuge again at 600G for 10 minutes.

Once the supernatant is removed, to identify endothelial cells within the heterogeneous SVF, incubate the cells with endothelial cell-specific CD31 or control IgG primary antibodies at four degrees Celsius overnight. Wash the cells three times with 0.1%PBST for 10 minutes at room temperature on a rocker to remove unbound primary antibody. And centrifuge at 600G for 10 minutes.

After discarding the supernatant, incubate the cells with a secondary antibody for one hour at room temperature on a rocker. Centrifuge at 600G for 10 minutes to remove secondary antibody staining solution. Wash the cells three times using PBST and then centrifuge as demonstrated earlier.

Re-suspend cells in the antifade mountant. Pipette the cell suspension onto microscope slides and cure overnight at room temperature to immobilize the cells. The next day, add 100 microliters of 100%glycerol.

Place a cover slip on top and seal it with nail polish. Place the prepared slide on the stage of a confocal microscope. In the microscope software interface, select the Light Path option and adjust the beam splitter setting to T90R10.

Next, verify the cutoff wavelength values for the secondary dichroic mirrors, or SDMs, in the Mirror setting. In the Detector menu, click on the Dye and Detector Select option to assign channels and excitation wavelengths. Then, set the laser power intensity percentage values for each specific laser in the range of 10 to 15%In the Series menu, under the LSM section, toggle the Z option to On.Set the Z step size to the system optimum of 0.74 micrometers.

Next, under the Z Section menu, enter the value for start input field and define a Z stack based on the membrane signal to capture the full cell volume. Under the Acquire panel, enter the file name and file save location in the LSM input field. And click on LSM Start to initiate image acquisition.

And save the files in oir native microscope format. After acquisition, use the Z navigation arrows to verify the signal and focal distribution of different dyes across the entire Z stack. Upload the saved oir file to Image J.In the Bio-Formats Import Options pop-up window, click Okay.

Click on Image Menu to open the file. And from the dropdown options, select Color, then Split Channels option to open the different channels as separate windows. Next, adjust the brightness contrast and optimize the LUT values using the Adjust option under the Image menu for enhanced visualization of the image.

Use the Z-axis scroll bar at the bottom of the frame to traverse the optical sections and verify cellular features throughout the stack. Under the Image menu, choose Stack. Then, select the Z Project function to generate summed intensity projections of the three-dimensional confocal image.

In the Z Projection dialogue box, define the start and stop slice ranges. Select the projection type and click Okay to obtain the final 2D composite image. Next, navigate to the Image menu.

Select the Type option to adjust the image type to 16-bit. Then, save and export the images as 16-bit TIF files. Import the saved images into CellProfiler version 4.2.8 for automated cell segmentation, quantification of the reflected light intensity for each cell, and exporting the intensity measurement values to a spreadsheet.

In the spreadsheet, define the threshold for senescence-associated beta-galactosidase positivity as the sum of average reflected light intensity of the pH 8 condition plus three standard deviations. Classify the cells in the pH 6 condition as senescence-associated beta-galactosidase positive if their intensity values exceed the established threshold. Reflected light senescence-associated beta-galactosidase staining of human SVF cells produced clear and reproducible patterns across three pH levels.

At pH 5, most cells showed a strong signal due to high endogenous enzyme activity. At the assay standard value of pH 6, the signal intensity and frequency decreased, allowing for the specific identification of senescence-associated beta-galactosidase cells. At pH 8, the signal was minimal, and this served as a baseline to define the threshold for positivity.

Detection sensitivity testing showed that the reflected light imaging outperformed Brightfield across all pH conditions. At pH 5, reflected light showed strong X-Gal precipitates, whereas Brightfield only caught the largest deposits. At pH 6, reflected light identified positive cells that were too weak or diffused to see in Brightfield.

At pH 8, reflected light imaging detected minimal signal, consistent with low enzymatic activity, outperforming Brightfield in identifying X-Gal precipitates at intermediate and lower activity levels. Altogether, this reflected light imaging workflow integrates the simultaneous detection of pH-dependent senescence-associated beta-galactosidase and fluorescence-based markers, such as CD31, facilitating multi-feature senescence assessments at single cell resolution within heterogeneous SVF populations. This protocol enables sensitive and quantitative SABG activity measurements while simultaneously allowing for both other senescence markers, but also other cellular markers.

The main challenge, I'd say, is the initial microscopy setup. But once that is optimized, you can use the same acquisition parameters across experiments. Future studies can combine this assay with other subcellular markers to see how SABG relates to different cellular processes.