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Created mammary gland fat and SVF separation protocol
Mammary Gland Fat and SVF Separation
* 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.
* 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.