Mammary Gland Fat and SVF Separation

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Warm PBS and the Erlenmeyer flask from the shaker in water bath at 37 degrees Celsius. Have an ice bucket ready.
Dissect mouse MG. Be consistent with the side and the size of the MG dissected. Be careful that the dissection is clean without skin, fur, blood vessels, or unwanted tissue.
Put the dissected MG in a petri dish with 1ml PBS to keep tissue moist.
Dump out additional PBS while holding the MG with forceps to ensure it does not fall. This ensures that the tissue will not become too soggy and hinder proper mincing.
Mince the tissue in the petri dish while maintaining a 30-45 degree angle. Mince using the bigger scissors for about 5-10 minutes until all the tissue chunks look smaller than 1mm.
Cut the tip of a 1000uL pipette’s tip. Using this tip, transfer out the minced tissue from the petri dish into a 50mL tube.
Add 1mL collagenase mix (BAT digestion mix) into the 50mL tube.
Seal the tube lid with parafilm. Place in the Erlenmeyer flask in the shaker at 37 degrees Celsius and 300 strokes for about 45-50 minutes or until the tissue looks digested and the pieces are not big anymore. Check the tube every 5-10 minutes to ensure digestion.
Once digestion in the shaker is over, pipette out the mixture into a 1.5mL tube. If the tissue is still big, cut the pipette tip’s tip to be able to pipette the tissue.
Centrifuge the 1.5mL tube at room temperature (24 degrees Celsius) for 5 minutes at 500 rcf/gauge.
Once centrifuging is done, the upper fat layer will be evident along with the chunky SVF lower layer as the pellet. The in-between layer solution is called the internatant.
Cut a 200mL pipette tip’s tip and pipette out the fat layer into a clean 1.5mL tube. Record the volume removed and then split the fat equally into 2 1.5mL tubes. For example, if you remove 800uL of fat (with internatant), then you will end up with 2 1.5mL tubes that have 400uL fat each. Put the fat tubes on ice.

Note: It is hard to isolate the fat while pipetting, so you should anticipate to pipette out some internatant with the fat.

Using a pipette, aspirate and dump the remaining internatant solution. Be careful not to pipette out the SVF pellet.
Add 600uL of PBS to the pellet tube and mix vigorously using the pipette (by pipetting repeatedly) to break down the pellet. Pipette 300mL SVF+PBS solution into a 1.5mL tube. Pipette 300mL SVF+PBs again into another 1.5mL tube. Put the SVF tubes on ice.
Dump the initial tube with the remaining SVF+PBS.
Freeze the 2 fat tubes and the 2 SVF tubes in the -80 freezer for later use.