13. Centrifuge at 4
C for 10 min at 10,000 Â g.
14. Pour supernatant into a 280 mL conical bottle, label with date,
volume, and tissue type and store at À20
C (see Note 60).
15. Resuspend pellet by vortexing in 5 mL/tube of 10 mM
Na 2 EDTA/0.1 M NaOH buffer and pour into a sterile
250 mL beaker.
16. Rinse tube(s) 3Â by vortexing and combine with resuspended
pellet. Adjust volume to ~200 mL and stir with stir bar on ice
for 2 h. Suspension may become viscous at this stage.
17. Pour suspension into the same 50 mL Oakridge centrifuge
tube and centrifuge at 10,000 Â g for 10 min at 4
C. Discard
supernatant as a biohazard.
18. Resuspend pellet by vortexing in 0.1 M Tris–HCl pH 7.2,
~15 mL/ tube, and pour into a sterile 250 mL beaker. Rinse
tubes twice by vortexing and combine in beaker (see Note 61).
Adjust the volume to 150 mL with Tris–HCL and then the
final volume to 200 mL with buffered water.
19. Weigh out 20 mg of collagenase per 30 g starting tissue and
dissolve in a small volume (~1 mL) of buffered water (see
Subheading 2.13, item 24). Sterile-filter through a 0.2 μm
Acrodisc syringe filter using a 1 mL syringe directly into the
beaker containing the resuspended pellet.
20. Add 60 μL of 1 M calcium chloride into beaker to activate the
enzyme. Weigh out Trypsin and Chymotrypsin (20 mg of each
per 30 g starting tissue) and dissolve in ~1 mL of buffered
water. Filter-sterilize through a 0.2 μm Acrodisc syringe filter
using a 1 mL syringe directly into the beaker containing the
resuspended pellet.
21. Cover and digest for an additional 2 h at 37
C.
22. Pour supernatant into the 50 mL Oakridge centrifuge tubes as
before, balance and place at 4
C for overnight storage.
23. Day 3. Centrifuge tubes at 4
C for 10 min at 10,000 Â g.
Discard supernatant into biohazard waste.
24. During centrifugation set up the separatory funnel. Place funnel at a 45
angle with a sterile beaker underneath it to prevent
leakage.
25. Shake 6% PEG/8% dextran/0.01 M phosphate saline buffer to
mix the two layers and resuspend the pellets in this buffer. Pour
suspension into a 400 mL beaker. Rinse tubes 2Â by vortexing
(see Note 61).
26. Adjust resuspension volume to 10 mL per gram of starting
tissue with 6% PEG/8% dextran/0.01 M phosphate saline
buffer.
44
Elizabeth Wallace et al.
C for 10 min at 10,000 Â g.
14. Pour supernatant into a 280 mL conical bottle, label with date,
volume, and tissue type and store at À20
C (see Note 60).
15. Resuspend pellet by vortexing in 5 mL/tube of 10 mM
Na 2 EDTA/0.1 M NaOH buffer and pour into a sterile
250 mL beaker.
16. Rinse tube(s) 3Â by vortexing and combine with resuspended
pellet. Adjust volume to ~200 mL and stir with stir bar on ice
for 2 h. Suspension may become viscous at this stage.
17. Pour suspension into the same 50 mL Oakridge centrifuge
tube and centrifuge at 10,000 Â g for 10 min at 4
C. Discard
supernatant as a biohazard.
18. Resuspend pellet by vortexing in 0.1 M Tris–HCl pH 7.2,
~15 mL/ tube, and pour into a sterile 250 mL beaker. Rinse
tubes twice by vortexing and combine in beaker (see Note 61).
Adjust the volume to 150 mL with Tris–HCL and then the
final volume to 200 mL with buffered water.
19. Weigh out 20 mg of collagenase per 30 g starting tissue and
dissolve in a small volume (~1 mL) of buffered water (see
Subheading 2.13, item 24). Sterile-filter through a 0.2 μm
Acrodisc syringe filter using a 1 mL syringe directly into the
beaker containing the resuspended pellet.
20. Add 60 μL of 1 M calcium chloride into beaker to activate the
enzyme. Weigh out Trypsin and Chymotrypsin (20 mg of each
per 30 g starting tissue) and dissolve in ~1 mL of buffered
water. Filter-sterilize through a 0.2 μm Acrodisc syringe filter
using a 1 mL syringe directly into the beaker containing the
resuspended pellet.
21. Cover and digest for an additional 2 h at 37
C.
22. Pour supernatant into the 50 mL Oakridge centrifuge tubes as
before, balance and place at 4
C for overnight storage.
23. Day 3. Centrifuge tubes at 4
C for 10 min at 10,000 Â g.
Discard supernatant into biohazard waste.
24. During centrifugation set up the separatory funnel. Place funnel at a 45
angle with a sterile beaker underneath it to prevent
leakage.
25. Shake 6% PEG/8% dextran/0.01 M phosphate saline buffer to
mix the two layers and resuspend the pellets in this buffer. Pour
suspension into a 400 mL beaker. Rinse tubes 2Â by vortexing
(see Note 61).
26. Adjust resuspension volume to 10 mL per gram of starting
tissue with 6% PEG/8% dextran/0.01 M phosphate saline
buffer.
44
Elizabeth Wallace et al.