Chondrocyte extraction

Buffer preparation

Collagenase solution

dissolve 200-250 mg of collagenase in 10 ml of DMEM4

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 vortex and filter (0.22 μm)

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to a 50 ml falcon tube add 44.5 ml DMEM4

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add 5 ml collagenase in DMEM4

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add 0.5 ml Pen/Strep

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add 1 ml FBS

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 make 1.5 ml aliquots and store at -20°C

DMEM 20

 40 ml DMEM4

 8 ml FBS

 0.5 ml Pen/Strep

Note: store at -20°C

Dissecting the ribs

skin and gut the mice (protocol for P0-P7)

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 remove rib cage

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remove the sternum, cutting as close as possible to where the ribs join the sternum

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cut the ribcage in two, cutting through the spine

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place in warm PBS to keep moist

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place samples from 3 mice (6 pieces) in 15ml falcon tubes with 1 aliquot of collagenase (1.5 ml)

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incubate at 37°C vortexing every 15 minutes

 .                         → 45 min (0-3 day old mice)
.                         → 1 h (3-5 day old mice)
.                         → 1 h 15 min (5-7 day old mice)

Dissecting the cartilage

place ribs in a 10mm Petri dish with PBS

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separate single ribs removing muscle and fibrous tissue

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isolate cartilage (translucent) and clean as much as possible

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place the dissected cartilage in a new Petri dish with PBS to keep moist

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transfer the dissected cartilage from 3 mice to an eppendorf tube with 1.5 ml of collagenase

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incubate at 37°C for 3-4 h, vortexing every 30 min

Chondrocyte extraction

 transfer cartilage in collagenase in a 50 ml falcon tube

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pipette up and down 5-10 times with a 5 ml stripette to disgregate cartilage

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pass through a cell strainer into a clean 50 ml falcon tube

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wash through with 15 ml of DMEM 20

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centrifuge at 1200 rpm for 5 min (350 x g)

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discard the supernatant (do not pour: pipette out!)

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wash the pellet with 15 ml of PBS

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centrifuge at 1200 rpm for 5 min (350 x g)

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discard the supernatant

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 proceed to RNA extraction or protein extraction

RNA isolation

Resuspend the pellet obtained from in 1 ml of TRIzol reagent, snap-freeze and store at -80°C until required for RNA isolation. Isolate RNA using the standard TRIzol protocol.

Protein prep

resuspend the cell pellet in 1.2 ml of PBS and count the number of cells/ml

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prepare aliquots of 10^5 cells in 1 ml

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centrifuge the aliquots at 9500 x g for 10 min

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resuspend pellets in 25µl of SDS-PAGE buffer with DTT (15µl dH2O, 5µl of 5 x SDS-PAGE buffer, 5µl 1M DTT)

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incubate at 95°C for 10 min

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freeze at -20°C until needed