- Add 20 ml of DMEM/F-12. Mix by pipetting up and down.
- Filtrate the mixture on a 70μm cell strainer (seeNote 8).
- Wash the 30 ml container with 10 ml of DMEM/F-12, and
pass it through the same cell strainer. - Centrifuge at 350gfor 10 min at 4C.
- Eliminate supernatant and lyse red cells with 1 ml of ACK
Buffer. Vortex until the solution become pink/red (3–5 s). - Stop lysis by adding 10 ml of FACS buffer.
- Centrifuge at 350gfor 7 min at 4C.
- Resuspend the pellet into 2 ml of FACS buffer.
- Distribute in polystyrene round bottom FACS tubes in order
to perform non-labeled, single labeling, CD34-FITC fluores-
cence minus one (FMO) and cell sorting tubes (seeNote 9). - Add 2 ml of FACS buffer in each tube.
- Centrifuge at 350gfor 5 min at 4C.
- Remove the supernatant, leave around 100μl and homogenize
the pellet. - Add 1 ml of the antibody mix (seeNote 10).
- Incubate the tubes protected from light for 20–40 min at 4C.
- Stop labeling by adding 2 ml of FACS buffer.
- Centrifuge at 350gfor 5 min at 4C.
- Resuspend the pellet with 0.5 ml of FACS buffer and filtrate
using a 30μm CellTrics strainer. - Wash the tube with 0.5 ml of FACS buffer and filter as above.
Keep the tubes on ice and protected from light until cell
sorting. - Just before sorting, add DAPI at final concentration of 1μg/
ml (seeNote 11). - To sort MuSC, please follow our gating strategy. Gate cells
based on their size and granularity, and then select single cell by
eliminating high FSC-H and SSC-H cells. To follow, gate on
negative cells for DAPI and CD45/CD31/Sca1. Finally,
MuSCs are the double-positive population for CD34 and
α7integrin (seeFig. 1). - Recover cells in polypropylene round bottom FACS tubes
containing 0.5 ml of FBS, and note the number of cells you
recovered for the cell culture part.
30.Quantum satisthe FACS tube with MuSC growth medium to
4 ml.
- Centrifuge at 350gfor 10 min at 4C.
- Discard carefully the supernatant.
- Resuspend the pellet in 1 ml of MuSC growth medium.
Muscle Stem Cells Fate 543