The overall goal of this procedure is to isolate adipose tissue immune cells. This is accomplished by first removing the epidermal fat pads. In the second step, the adipose tissue is digested with collagenase.
Next, the adipose tissue stromal vasculature fraction is isolated via differential centrifugation. In the final step, the adipose tissue immune cells are labeled with fluoro four conjugated primary antibodies. Ultimately, the proportion of viable macrophages and T cells residing in the adipose tissue can be quantified by flow cytometry.
Hi, I'm Alyssa Hasty. Today we will show you how to isolate and quantify immune cells from adipose tissue. This method can help answer key questions in the immuno metabolism field, such as how immune cell populations in the adipose tissue change following genetic pharmacologic or nutritional manipulation.
Demonstrating the procedure today will be Arian Kennedy and Jeb, or postdoc fellows in my lab. After thoroughly wetting the fur of the euthanized animal with 70%ethanol, make an incision at the level of the xiphoid process and open the thoracic cavity to expose the heart taking care not to sever any major blood vessels. Next, clip the right atrium to allow the blood and perfu eight to exit the circulatory system.
Then use a pair of forceps to grasp the heart and gently insert a needle through the apex into the left ventricle. Now slowly perfuse the mouse with 15 milliliters of sterile PBS. Then open the peritoneal cavity and remove the pereg gonadal fat pads using care to avoid any gonadal tissue.
Place the fat pads in a whey boat on ice containing two milliliters of magnesium and calcium free DPBS supplemented with 0.5%BSA, and then mince the adipose tissue into fine pieces. This is what the fat should look like upon completion of mincing. Now pour the minced adipose tissue homogenate into a 15 milliliter conical tube.
Rinse the webo with one milliliter of DPBS supplemented with BSA and pour the wash into the conical tube. Next, rinse the WHE boat with three milliliters of collagenase solution and pour the wash into the tube. Incubate the homogenous in a rotational shaker for 20 minutes at 200 RPM and 37 degrees Celsius.
Then add 10 milliliters of BSA supplemented DPBS to the homogenate using a 10 milliliter serological pipette. Next, iterate the digested homogenate several times and place the tube on ice. Pass the cell suspension through a 100 micrometer filter into a new 50 milliliter conical tube.
After spinning down the cells, decant the snat and then lice the contaminating erythrocytes within the stromal vascular fraction cell pellet. With three milliliters of a CK buffer. Quickly add fax buffer to neutralize the A CK buffer.
After washing and repre pelleting the cells resuspend the pellet in fax buffer. Then count the viable cells by trian blue exclusion and further dilute the cell suspension to a final concentration of five to 10 times 10 to the six cells per milliliter. Begin the staining by incubating the cells with anti-US CD 16, CD 32 FC blocking antibody to a final concentration of 0.5 to one microgram per 10 to the six cells for 10 minutes on ice.
Then divide the cells into greater than or equal to 10 to the six aliquots and transfer the aliquots into 12 by 75 millimeter polystyrene round bottom tubes. Combine any extra cells in an adequate number of tubes to accommodate the required compensation and modified fluorescence minus one controls. Then add fluoro four conjugated primary antibodies and isotype controls to the appropriate tubes at the appropriate concentrations.
After incubating the samples in the dark at four degrees Celsius for 30 minutes, wash the samples twice in two milliliters of fax buffer Resus suspending the pellets in greater than or equal to 400 microliters of fax buffer After the second wash, then transfer the samples to new 12 by 75 millimeter polystyrene round bottom tubes equipped with 35 micrometer cell strainer tube tops. Just before analysis, add a viability die for live dead cell discrimination to the appropriate samples. Then use an unstained negative control to adjust the side scatter on a log scale and the forward scatter on a linear scale.
Next, draw an initial light scatter gate based on the type of cell being analyzed and adjust the photomultiplier tube gain so that the unstained cells are on the far left of a single parameter histogram approximately centered on the tend of the two mark for the appropriate channels. Using antibody capture compensation beads, perform the multicolor compensation and then set the experimental gates based on the previously prepared modified fluorescence minus one controls. Finally, collect the appropriate number of events based on the prevalence of the population of interest and record the experimental data.
In this first series of density plots, stromal vascular fraction cells isolated from urine epidermal adipose tissue were stained for F four 80 and CD 11 B for the identification of adipose tissue macrophages. These first three panels demonstrate the initial gating strategy, including the light scatter doublet discrimination, and viability gates. In these next two panels, representative modified fluorescence minus one controls used to set the experimental gates can be observed.
In this example, the F four 80 and CD 11 B antibodies were replaced with appropriate isotype controls to account for autofluorescence and non-specific binding. The quadrant gates were then drawn to identify the viable F four 80 CD 11 B positive adipose tissue macrophages. The proportion of F four 80 positive CD 11 B positive adipose tissue macrophages isolated from low fat and high fat fed mice could then be compared as depicted in these representative density plots.
In this next series of panels, the initial light scatter gate was restricted to the lymphocyte population in order to avoid including any autofluorescent myeloid cells. In these last two pairs of panels, representative gating for CD four positive and CD eight positive adipose tissue T cells is shown. Viable adipose tissue lymphocytes were first gated for TCR R beta tcr.
R beta positive adipose tissue T cells were then gated for CD four and CD eight expression. The proportion of TCR beta positive T cells and of CD four positive and CD eight positive T cells isolated from the adipose tissue of high fat and low fat fed mice could then be compared. After watching this video, you should have a good understanding of how to isolate adipose tissue immune cells.
