Immunomagnetic Separation of Fat Depot-specific Sca1^high Adipose-derived Stem Cells (ASCs)

Ethics Statement: The University of Michigan Committee on Use and Care of Animals (UCUCA) has approved all methods and protocols in accordance with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council). Mice are maintained in a University of Michigan vivarium and are given free access to food and water and kept on a 12 hr dark/light cycle.

1. Preparations

1. Prepare primary culture media with DMEM, 10% FBS, 1x P/S/G, and 1x antibiotics/antifungals. This will be used to keep tissues in before digestion. Place stock and aliquot in 37 ºC water bath.
2. Prepare five milliliters of Type III collagenase solution at 5mg/ml dissolved in HBSS (+Ca, +Mg) for each type of fat pad being isolated, up to five mice. For example, when isolating SQ and VIS from a single genotype, make 10 ml, if wild-type and mutant SQ and VIS, make 20 ml. Adjust pH to 7.4 and sterile filter. Aliquot into 50 ml tubes. Set aside away from light.
3. Use 70% ethanol to disinfect surgical field. Wipe down and cover with a blue pad. Spray some ethanol on the blue pad.
4. Use the 22 G needles to pin down an absorbent pad to the styrofoam board and trim with scissors. Spray the pad with ethanol then set the board on the blue pad and cover with another blue pad until beginning dissection.
5. Fill two 50 ml tubes with ethanol and place in a stand adjacent to the surgical field. Place the scissors and forceps in the ethanol.
6. Fill another 50 ml tube with PBS plus 1x anti-anti for rinsing ethanol off surgical tools.
7. In the hood, label 60 mm dishes for each tissue type and fill with culture media warmed to 37 ºC. Place the plates adjacent to the surgical area.

2. Isolation of Subcutaneous (SQ) Fat Pads

1. Euthanize mouse with an overdose of isoflurane and pneumothorax.
2. Gently spray mouse with 70% ethanol and lay supine. Pin the paws to the foam board with 22 G needles.
3. Make a small cut in the skin of the lower abdomen. Holding the top of the cut with the forceps, take the scissors and separate the skin from the peritoneum.
4. Once the skin is separated from the peritoneum from the abdomen to the thorax, reflect the skin away from the peritoneum towards the head by making lateral cuts in the skin along the side of the body.
5. Reflect the remaining skin holding the inguinal fat away from the body and pin down to the board with 22 G needles.
6. Switch to fine scissors and forceps. Grab the inguinal fat pad with the forceps at the origin near the peritoneum and snip away between the skin and inguinal fat progressing towards the groin avoiding contaminating the SQ with skin.
7. Place the insolated inguinal fat pad in the labeled 60 mm dish.

3. Isolation of Visceral (VIS) Fat Pads

1. In a similar fashion to step 2.5, cut the peritoneum open to expose the visceral fat pads and gut.
2. Move the gut away toward the thorax.
3. Grab the VIS fat pad at the distal end and gently pull up. Dissect out the VIS fat pad while being careful to exclude epididymal (or uterine, if using female mice) tissue.
4. Place in labeled dish and repeat step 3.3 for the remaining VIS pad.

4. Collagenase Digestion of Fat Pads

1. Move 60 mm dishes to the tissue culture hood and aspirate off media.
2. Mince the fat pads with curved scissors then add to collagenase solution. Be sure to rinse the scissors and forceps in between tissue types with ethanol and PBS.
3. Shake at 300 rpm and 37 ºC for 10-20 min until tissues are digested. It is acceptable if some pieces of SQ are still visible. This will be addressed in a later step.
4. Add 25 ml of culture media to the collagenase solution to stop the collagenase treatment, and pipette up and down 10 times with a 10 ml serological pipette to disperse the digested tissues.
5. Strain cells using a 100 µm cell strainer. Centrifuge for 10 min at 300 x g.
6. Carefully decant off media not to disturb the pellet and add 5 ml of sterile water to the pellet and gently pipette to suspend cells. Wait 2 min to lyse erythrocytes.
7. Add 25 ml of media with 10% FBS and strain cells through a new 100 µm cell strainer.
8. Centrifuge for 10 min at 300 x g. Decant media and resuspend pellet in 1 ml of culture media.
9. Count cells with a hemocytometer using 1:1 trypan blue.
10. Plate 1 x 10⁶ cells in one well on a 6-well plate.

5. Magnetic Cell Separation

1. After about 4 to 6 hr of cell adhesion, rinse cells twice with HBSS (-Ca, -Mg). Dissociate adherent cells using 0.05% trypsin. Dilute trypsin cell suspension in 1 ml separation buffer and spin for 5 min at 300 x g.
2. Aspirate supernatant. Resuspend cells in 500 µl buffer. Remove a 10 µl aliquot and count cells using a hemocytometer.
3. Spin cells for 5 min at 300 x g.
4. Aspirate supernatant completely. Resuspend cells in 90 µl buffer. Add 10 µl anti-Sca1-FITC primary antibody. Mix well and incubate for 10 min at 4 ºC in the dark.
5. Wash cells with 1 ml buffer and centrifuge cells for 5 min at 300 x g. Remove supernatant completely.
6. Resuspend cells in 80 µl buffer. Add 20 µl anti-FITC microbeads. Mix well and incubate for 15 min at 4 ºC in the dark.
7. Wash cells with 1 ml buffer and centrifuge cells for 5 min at 300 x g.
8. Remove supernatant and resuspend pellet in 500 µl buffer.
9. Place column in the magnetic holder and rinse column with 500 µl buffer.
10. Add cell suspension to column. Avoid bubbles while pipetting.
11. Collect the unlabeled Sca1^– cells and wash column 3 times with 500 µl buffer. Only add new buffer when the reservoir is empty. Avoid bubbles while pipetting.
12. Remove column from the magnetic holder and place into a 15 ml conical tube.
13. Add 1 ml buffer to column and elute Sca1⁺ by pushing supplied plunger through the column reservoir.
14. Spin down cell fractions for 5 min at 300 x g. Resuspend cells in 1 ml culture media.
15. Remove a 10 µl aliquot from the labeled and unlabeled fractions and count cells using a hemocytometer.
16. Plate each cell fraction in 1 well of a 6-well plate.

6. Verification of Immunomagnetic Separation of Sca1^high ACSs with Flow Cytometry

1. Rinse cells twice with HBSS (-Ca, -Mg) and dissociate cells using 0.05% trypsin.
2. Centrifuge cells at 300 x g for 5 min. Resuspend cells in 1 ml culture media and count using a hemocytometer.
3. Obtain >10⁶ cells in 1 ml culture media and centrifuge at 300 x g for 5 min.
4. Remove supernatant and repeat step 6.3 two more times.
5. Resuspend cells with 1 ml of 2% goat serum + 2% BSA and block for 30 min at RT.
6. Centrifuge cells at 300 x g for 5 min.
7. Remove blocking solution.
8. Add rat IgG2a Alexa Fluor 647 (0.25 µg, 1:400) or anti-Sca1 Alexa Fluor 647 (0.25 µg, 1:400), in 100 µl of PBS with 2% goat serum and 2% BSA + PBS at 4 °C for 30 min in the dark.
9. Add 1 ml of cold PBS to cells. Centrifuge 300 x g for 5 min at 4 °C .
10. Remove supernatant and repeat step 6.9 two more times.
11. Resuspend cells in 1 ml PBS. Pass cell suspensions through a 100 µm cell strainer in preparation for flow cytometric analysis.