2.3

Extraction of RNA

for Validation of the

System

1. TRIzol reagent.

2. PBS.

3. Clean scissors: autoclave before use.

4. Clean tweezers: autoclave before use.

5. A 10 mL tissue grinder: include a mortar and a pestle; autoclave

before use.

6. PureLink™RNA Mini Kit.

7. Liquid nitrogen.

8. Ethanol: Molecular Biology grade.

9. 70% ethanol in RNase-free water: Molecular Biology grade.

10. Chloroform: Molecular Biology grade.

11. 2 mL RNase-free tubes.

3

Methods

Carry out all procedures at room temperature unless otherwise

specified. All medium should be prewarmed to 37
C before adding

to cells.

3.1

Isolation of Mice

TDSCs

1. Euthanize the 6–8-week-old C57BL/6 mice by cervical dislo-

cation. Shave the hair and sterilize hind limbs with 70% ethanol

spray.

2. Isolate the hind limb at femur head level. Remove the sur-

rounding adipose tissue and blunted isolation of connective

tissue to expose patellar tendon and Achilles tendon (see Note

1).

3. Isolate the mid portion of Patellar and Achilles tendon and

immediately rinse them by wash buffer. Isolated tendon tissue

was homogenized and digested by 3 mg/mL Type 1 collage-

nase for 3 h (see Note 2).

4. Filter the digested tendon remains by 70 μm cell strainer, and

remove residual tendon fibers.

5. After centrifuging, culture the isolated cells with complete

medium in T-25 flask. Incubation conditions are 37
C in a

humidified atmosphere and containing 5% CO2.

6. Make a single cell suspension at 10⁷ cells/mL in FACS buffer

(see Note 3).

7. For each staining sample, put 100 μL of 10⁶ cells into a sorter

tube. For each antibody combination, add conjugated antibo-

dies (CD44, CD45, CD90, and CD34) to the samples at final

concentration of 10 μg/mL, and incubate for 30 min in dark at

138

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