Digestion of Mouse Skeletal and Cardiac Muscles for Fluorescence-Activated Cell Sorting and Culture of Fibro-Adipogenic Progenitors (FAPs)

To begin, add two milliliters of digestion buffer to a five milliliter transport vial containing the mouse heart tissue, and four milliliters of digestion buffer into a 15 milliliter centrifuge tube containing the mouse skeletal tissue. Place the heart and quadricep muscle tissue isolated from a spiny mouse onto the lid of a Petri dish. With a pair of tissue scissors, mince it into pieces smaller than one cubic millimeter.

Transfer the minced tissue into the respective digestion tubes. Vortex the contents of the tubes for two seconds at low speed. After 20 minutes, remove the tubes off the rotator.

Allow the tissue pieces to settle. Use a P 1000 pipette to aspirate the supernatant. Transfer the supernatant into 50 milliliter centrifuge tubes containing 20 milliliters of ice cold FACS buffer.

Top up the digestion tubes with the respective volumes of digestion buffer. Incubate them on the rotator again. After subjecting the muscles to another round of digestion, add ice cold FACS buffer to quench the digestion.

Place a 40 micrometer cell strainer on top of a 50 milliliter centrifuge tube. Filter the digestion suspension and supinate through the strainer. Centrifuge the cell suspension at 500 G for eight minutes at four degrees Celsius.

After removing the supernatant, add three milliliters of ACK lysis buffer to the pellet and resuspend. Incubate the suspension for five minutes on ice. Top up the volume to 40 milliliters with FACS buffer.

Centrifuge the samples again at 500 G for five minutes at four degrees celsius. After decanting the supernatant resuspend the pellet in 0.5 milliliters of FACS buffer. Finally, filter the suspension through a 40 micrometer cell strainer cap, placed on a five milliliter polystyrene round bottom tube.