The protocol allows investigators to isolate functionally and molecularly distinct subpopulations of perigonadal white adipose tissue stromal cells from mouse models of interest. The advantage of this technique is that all tools and reagents are widely available. Demonstrating the procedure will be Lavanya Vishvanath, a senior laboratory research analyst from Dr.Rana Gupta's laboratory.
After stromal vascular cell isolation from gonadal white adipose tissue, or WAT, resuspend the cell pellet in one milliliter of red blood cell lysis buffer for one to two minute incubation at room temperature. Stop the lysis with 10 milliliters of 2%FBS and PBS and filter the cells through a 40 micron cell strainer into a new 50 milliliter centrifuge tube. Collect the cells by centrifugation and resuspend the pellet in 400 to 800 microliters FBS and PBS, supplemented with FC block.
After a 10 minute incubation at four degrees Celsius, transfer 400 to 800 microliters of cells to the appropriate number of tubes for fluorescence-conjugated antibody staining and label the cells with the control and experimental antibodies of interest at four degrees Celsius for 15 minutes, protected from light. At the end of the incubation, pellet the cells by centrifugation and wash the cells in 400 microliters of fresh FBS and PBS per tube. After the wash, resuspend the pellets in 400 to 800 microliters of fresh FBS and PBS per tube, and filter the cells through 40 micron filter caps into five milliliter polystyrene round bottom tubes for FACS.
Set the compensation and experimental gates to obtain the antigen-presenting cells and fibroinflammatory progenitors. After cell sorting, collect and wash the cells by centrifugation, and resuspend the pellets in 500 microliters of gonadal antigen-presenting cell culture medium per fraction. For immunomagnetic bead separation of the adipogenic and non-adipogenic cell fractions, resuspend the stromal vascular pellet in 10 milliliters of MS buffer and filter the cells through a 40 micron strainer.
After counting, collect the cells by centrifugation and resuspend the cells at no more than one times 10 to the eighth cells per milliliter of MS buffer concentration. Next, add no more than 0.25 grams of anti-CD31 and anti-CD45 biotin-conjugated antibody per one times ten to the six cells for a 15 minute incubation on ice. At the end of the incubation, wash the cells in four milliliters of MS buffer by centrifugation, and resuspend the pellet in 100 microliters of fresh MS buffer.
Add 10 microliters of streptavidin nanobeads to the cells for a 15 minute incubation on ice, followed by another wash in four milliliters of MS buffer. Resuspend the pellet in 2.5 milliliters of fresh MS buffer and transfer the cells to a five milliliter polypropylene tube. Place the tube onto a magnet for five minutes before decanting the unlabeled cells into a new 15 milliliter centrifuge tube.
Repeat the wash two more times as demonstrated, pulling the unlabeled cells after each wash, and collect the endothelial and hematopoietic lineage-depleted cells by centrifugation. Resuspend the pellet in 100 microliters MS buffer and label the cells with no more than 0.25 micrograms of Ly6C and CD-9 antibodies per one times 10 of the sixth cells for 15 minutes on ice. At the end of the incubation, collect the unbound Ly6C negative CD-9 negative adipogenic cells as just demonstrated before collecting the Ly6C positive non-adipogenic cells.
Then, collect both cell populations by centrifugation and resuspend the cells in 500 microliters of gonadal antigen-presenting cell culture medium per fraction. To set up cell cultures for differentiation, plate each fraction at four times 10 of the fourth cells per well in concentration in individual wells of a 48 well culture plate and place the plates in the cell culture incubator for seven to 10 days. The adipocyte precursor cells will begin to undergo differentiation into adipocytes as they approach confluence, while the fibroin inflammatory progenitors will be resistant to undergoing adipogenesis.
Using this protocol, distinct stromal cell populations can be isolated from intraabdominal WAT depots of adult mice by FACS or immunomagnetic bead separation as demonstrated. With immunomagnetic bead separation, adipogenic and non-adipogenic cells can be isolated from the gonadal WAT stromal vascular fraction. As observed by flow cytometry, 75%of the cells within the adipogenic fraction are Ly6C negative CD-9 negative adipocyte precursor cells, while over 75%of the non-adipogenic fraction, Ly6C positive fibroinflammatory progenitor cells.
Adipocyte precursor cells isolated either method differentiate into lipid-containing adipocytes to a high purity within seven to 10 days of plating. In contrast, non adipogenic precursors remain fibroblast-like and do not become adipocytes under the same culture conditions. The protocol works for the isolation of adipogenic and fibroinflammatory stromal cell subpopulations from perigonadal white adipose tissue.
Similar cell populations can be found in other adipose depots of mice. However, their selection will rely on the use of different antibodies. The cell populations isolated through FACS and magnetic separation can be used for in vitro assays of cell differentiation or further characterized through gene expression analysis.
