The overall goal of this procedure is to visualize and quantify the differentiation of stromal cells into adipogenic lineage, using a lineage-specific protein marker, fatty acid binding protein four. This is accomplished by first isolating and expanding the stromal cells of interest. The stromal cells are then harvested and plated into standard in-vitro culture plates.

After incubation for a few days, cells are treated with an adipogenic differentiation medium. Cells are incubated for 14 days, with regular media replacement. After differentiation, cells are fixed and stained with antibodies against fatty acid binding protein four.

An automated high-content screening immunoflourescent microscope is used to capture images of the labeled cells in the microwell plates. The differentiation is then quantified, using specialized image analysis software. In contrast to the traditional methods of measuring adipogenic differentiation utilizing dyes such as Oil Red O, the assay described in this video utilizes an antibody against lineage-specific protein, fatty acid binding protein four, to confirm adipogenic differentiation.

In doing so, this method quantifies changes in gene expression that correspond to adipogenic differentiation. This assay is able to deliver a wealth of information, including percentage of differentiated cells, intensity of the fluorescence signal per cell, and changes in cell morphology. The ability to analyze multiple characteristics enables the identification of potential subtle changes in differentiation in response to various treatments.

This assay also has high through-pull capability, making it ideal for high through-pull drug screening applications. Resuspend cells in complete ASC medium, which is DMEM/F-12 basal media, supplemented with 10%fetal bovine serum, GlutaMAX, and antibiotics. Pipette into a 96-well culture plate, at 5000 cells per well.

Eight wells are needed for each donor. Four wells receive control medium, while the rest receive differentiation medium. Every fourth well of each treatment will serve a node primary control.

Incubate cells at 37 degrees, humidified with an atmospheric condition of 5%carbon dioxide for four days. This is to allow cells to grow to confluence in each well. At day four, take the plates out of the incubator.

Remove half of the media from each well. Add equal volume of complete ASC medium, or adipogenic differentiation medium, which is supplemented with insulin, isobutylmethylxanthine, dexamethasone, and indomethasone. Incubate plate at 37 degrees, humidified with an atmospheric condition of 5%carbon dioxide.

The period of time required for sufficient differentiation is 14 days. During this time, replenish media by performing media change every two to three days. After differentiation, take plates out of the incubator.

Carefully pipette all media out of each well. Fix cells by adding ice-cold methanol. Incubate for five minutes at room temperature.

Remove the methanol, then wash with Tris-buffered saline. Block by adding 0.25%of casein blocker. Incubate for 10 minutes at room temperature.

Remove casein, then wash with Tris-buffered saline. Prepare primary antibody mix by diluting anti-FABP4 antibody 200-fold in antibody dilution buffer. Add mixture to all wells, except for those reserved as node primary control.

Add antibody dilution buffer to these wells instead. Incubate at room temperature for one hour. After incubation, wash cells once with Tris-buffered saline.

Remove, add fresh Tris-buffered saline, and incubate for five minutes on the rocker. Repeat this process twice. Prepare second reantibody mix by diluting Anti-Rabbit Alexa 488 second reantibody 200-fold in antibody dilution buffer.

Add DAPI, which labels cell nuclei, at a final dilution of one to 2000. Add the mixture to all the wells, and wrap plate with foil to prevent photobleaching. Incubate at room temperature for 30 minutes.

After incubation, wash cells with TBS, and further two 15-minute washes on the rocker. Remove all buffer from wells and add storage buffer, supplemented with 0.4 milligrams per milliliter thimerosal, to prevent bacterial growth. A high-content screening machine equipped with automated stage, focusing objectives, and filters, is used to image this bioassay.

Check the correct filters are in place. Clean the bottom of the plate for best quality. Load the plate into the stage with A1 positioned on upper-left-hand corner.

Using the plate acquisition wizard, record essential information about the experiment, such as plate number, magnification, date, experimental conditions, and labeling details. Select the type of microwell plate used. Users are able to select wells and the number of sites to be acquired.

Select all wells to be imaged by highlighting the grid. Move the stage to the brightest well. Selecting the brightest well will ensure that all images will be acquired with pixel-gray values that are in a linear range, and therefore directly proportional to staining intensity.

If this step is not performed, then images taken in brighter wells may be oversaturated. Select the appropriate channel combinations, exposure time, and Z-offset parameters for the experiment. The filters used for imaging of FABP4 and DAPI correspond to the specific labels used to visualize staining.

Alexa 488, which excites at 480 nanometers and emits at 560 nanometers, was used to visualize FABP4. And the DAPI dye, which excites at 360 nanometers and emits at 460 nanometers, was used to visualize cell nuclei. For plates labeled with Oil Red O, fat droplets were visualized with bright fuel-transmitted light.

And the Oil Red O label was also visualized with flourescence, as it excites at 575 nanometers and emits at 630 nanometers. When all the setup is complete, the biological assay is ready to be acquired. The image is automatically saved to the online server, to enable easy remote access to images.

The complete set of images stored on the online server are accessible using the remote desktop program. The images can then be analyzed using the MetaXpress software. The cell scoring application allows users to select a wavelength for detecting all nuclei in a field, such as DAPI, in this instance.

Subsequent wavelengths can be used to detect a positive marker in either the nucleus, cytoplasm, or both cell locations. Fatty acid binding protein four, or FABP4, is a marker of adipogenic lineage. The expression of FABP4 is localized to the nucleus and cytoplasm of differentiated adipocytes.

In this biological assay, we analyze FABP4 expression, using the cell scoring module. First, detect cell nuclei using DAPI channel, with user-defined parameters for size and signal intensity above background. Detect FABP4 signal using foot C channel, and user-defined parameters for size and signal intensity above background.

In this biological assay, Oil-Red-O-labeled fat droplets, which flouresce in the 560-nanometer range, were analyzed with a MetaXpress can module, Transflour. This analysis algorithm detects total cells using DAPI-labeled nuclei, which meet the cell size and staining intensity criteria specified by the user. The user also specifies the size and intensity of pits, which in this instance detect fat droplets in close vicinity to cellular nuclei.

Information regarding fat droplet size, staining intensity, number per cell, are logged by the Transflour assay. It's important to note that the Oil Red O label did leak out, or dissolve out of some cells, potentially limiting and confounding the true amount of differentiation taking place. Representative images of early and late passage, control, and differentiated cells, labeled with DAPI, a nuclear stain, and FABP4 are shown alongside merge and segmentation images.

The segmentation images display differentiated cells with a green overlay and undifferentiated cells with a red overlay. The percentage of cells expressing FABP4 was used as a measure of adipogenic differentiation potential. A significant increase in FABP4-expressing cells was observed with adipogenic differentiation in early and late passage cells.

Early passage cells demonstrated significantly greater increase in differentiation than late passage cells. The representative images of DAPI and Oil Red O are shown alongside merge and segmentation images. The segmentation images depict all nuclei with green overlay, and Oil-Red-O-stained lipid droplets with a red overlay.

The area of Oil Red O staining per cell was used as a measure of adipogenic differentiation potential. A significant increase in Oil Red O staining area was observed with adipogenic differentiation. Early passage cells showed greater area of Oil Red O stain per cell, compared to late passage cells.

Western blot was performed to analyze FABP4 protein expression level of differentiated early and late passage ASCs. Semi-quantitative analysis of the optical density of FABP4 bands as a ratio of total protein loading control showed that FABP4 immunolabeling was significantly increased in early passage, and to a lesser degree, late passage differentiated cells. By watching this video, you should have gained an understanding of how to differentiate stem cells into adipogenic lineage, how to perform immunoflourescence staining using adipogenic gene-specific marker, fatty acid binding protein four, and finally, how to image and analyze all relevant morphological features from cells that are immunopositive for fatty acid binding protein four.

I hope you find this video useful for your research.