Our research group is trying to understand the cell composition and cell specific transcriptional profiles of intermuscular adipose tissue or IMAT, in humans. IMAT, is actually an ectopic adipose depot residing between and around muscle fibers. Computed tomography and M-R-I have been able to quantify IMAT volume and have recently shown that it increases with both age and B-M-I, and is also associated with muscle degenerative diseases.
In vitro secretome studies have recently shown that IMAT secreted proteins have the ability to influence insulin sensitivity, inflammatory profiles, and also contractile protein expressions in human skeletal muscle. The limited amount of IMAT tissue collected during biopsies and the lipid laden nature of adipocytes make this tissue incompatible with tissue digestion and single cell RNA-Seq. Therefore, we wanted to develop a protocol that performed nuclei isolation on small amounts of tissue that could then be used in downstream single nuclei RNA-Seq.
This protocol will enable researchers to determine the compositional and cell-specific transcriptional alterations that occur at IMAT, in different metabolic disease states, and in response to interventions. We hope that this information will be crucial in developing new therapeutic strategies to help alleviate metabolic disease by either targeting IMAT directly or its secreted factors. The field severely lacks data on the biology of IMAT, its cellular origins, and communication with other cells, resident both within and outside of IMAT, as well as its molecular responses to lifestyle interventions, such as diet and exercise, or pharmacotherapy.
All of these are key research questions for our group. To begin, fill the canister with liquid nitrogen. Place mortars, pestles, micro scoops, spatula, glass Dounce, stainless steel pestle, and automatic Douncer on the working platform.
Pre-cool the glass Dounce in a beaker filled with ice. Fill the two mortars containing the pestle and spatula with liquid nitrogen to cool down the instruments. Meanwhile, add one milliliter of homogenization buffer to the glass Dounce.
Fill both mortars with liquid nitrogen and pour the 50 milligrams of human intermuscular adipose tissue sample into one of the mortars. For pulverization, gently press the pestle on the piece of tissue to break it into small pieces. Once the tissue is pulverized and there is still 1/4 to 1/2 of the mortar of liquid nitrogen left.
Tilt the mortar, collecting pulverized tissue in the mortar's lip. Once the liquid nitrogen evaporates, scoop the pulverized tissue into the glass Dounce containing homogenization buffer. On the automatic Douncer, move the glass Dounce up and down the stainless steel pestle for 10 strokes in the forward direction, followed by 10 strokes in the reverse direction.
Once the solution is cloudy, and free from any visible tissue fragments, transfer it to a pre cooled 1.7 milliliter LoBind tube on ice. Rinse the Dounce with 400 microliters of homogenization buffer, and transfer it to a LoBind tube. After collecting homogenized human intermuscular adipose tissue into a 1.7 milliliter LoBind tube on ice, add 14 microliters of 10%Triton X-100 to the homogenate.
Incubate the tube in dark on ice for 10 to 15 minutes while vortexing every three minutes. In a separate 50 milliliter conical tube, pre-wet 1/100 and 1/40 micron cell strainer with 100 microliters of D-P-B-S. Filter the homogenate through to 100 micron cell strainer.
Rinse a 1.7 milliliter LoBind tube with 400 microliters of homogenization buffer, and filter the suspension through the 100 micron cell strainer. Next, filter the suspension obtained through 100 micron filtrate on a 40 micron cell strainer. Evenly distribute the solution into two pre cooled 1.7 milliliter LoBind tubes.
Centrifuge the tubes at 2, 700 G for 10 minutes at four degrees Celsius. Discard the top lipid layer, and supernatant leaving approximately 50 microliters in the first tube. Gently pipette up and down 20 times without creating bubbles to resuspend the pellet in the first tube and transfer to suspension to a new 1.7 milliliter LoBind tube.
Next, add 500 microliters of nuclei isolation medium to the 1.7 milliliter LoBind tube and mix with pipetting. Centrifuge the tube at 1000 G for 10 minutes at four degrees Celsius. Remove the supernatant, leaving approximately 50 microliters in the tube and pipette gently to resuspend the pellet.
Then, add 200 microliters of nuclei isolation medium, and mix the suspension. Add one drop of the live cell staining solution and incubate in the dark on ice. After 15 minutes, filter the solution through a 30 micron cell strainer.
Mix the nuclei solution and add 10 microliters to a cell counting chamber slide. Count the nuclei using an automated cell counter.
