Mammary Gland Fat and SVF Separation: Difference between revisions
Created mammary gland fat and SVF separation protocol |
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* Centrifuge the 1.5mL tube at room temperature (24 degrees Celsius) for 5 minutes at 500 rcf/gauge. | * 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. | * 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. | * 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. | 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. | * Using a pipette, aspirate and dump the remaining internatant solution. Be careful not to pipette out the SVF pellet. | ||