Solutions for Tissue/Cell Digestion

Worthington 3x crystallized trypsin (LS03707), 1.3 mg.

Sterile Saline G (pH 7.0), 100 ml.

Procedure (all steps on ice)

1. Dissolve 1.3 mg trypsin in 100 ml Saline G.

2. Sterile filter into several 100 ml medium bottles, ~25 ml/bottle.

3. Store at -20^oC.

0.125% Trypsin with 0.025% Collagenase

Materials

Trypsin 1:300 (GIBCO 840-7073) 62.5 mg Collagenase (Sigma C-0130) 12.5 mg Glucose 50 mg or 0.5 ml of 10% Saline D (20x) 2.5 ml

distilled H2O to a final volume of 50 ml Procedure

1. Mix components as shown in Table.

2. Filter first through Whatman #1 filter paper, then through a Millipore 0.22 um filter.

3. Dispense into 15 ml conical disposable tubes, 5 ml each.

4. Store at -20 or -70^oC.

Trypsin 0.05%

Materials

Trypsin 1:300 (GIBCO 840-7073) 0.5 g Glucose 1.0 g Saline D (20X) 50 ml or 1x 1000 ml

Procedure

1. Mix comoponents as shown in Table, bring up volume to 1000 ml.

2. Filter first through Whatman #1 filter paper, then through a Millipore 0.22 um filter in hood.

3. Dispense into 250 ml media bottles, 100 ml (10 bottles) each.

4. Store at -20 for daily use and -70^oC for long term.

Trypsin 0.25%

Materials

Trypsin 1:300 (GIBCO 840-7073) 2.5 g TD 1000 ml

Procedure

1. Dissolve trypsin in TD.

2. Filter first through 2 layers of Whatman #1 filter paper, then through a Millipore 0.22 um belljar filter. Rinse out the pump tubing thoroughly.

3. Dispense into 250 ml sterile media bottles, 100 ml each.

4. Store at -20 for daily use and -70^oC for long term.

Saline D (20X)

500 ml 100 ml Phenol red 12 mg or 12 ml of 0.1% 2.4 mg or 2.4 ml of 0.1% NaCl 80.0 g 16.0 g KCl 4.0 g or 18 ml of 3 M 0.8 g or 3.6 ml of 3 M Na2HPO4 0.45 g (7H2O) 91 mg (7H2O) or 17 ml of 100 mM 3.4 ml of 100 mM KH2PO4 0.3 g 60 mg or 22 ml of 100 mM 4.4 mlof 100 mM Saline D (1X)

1000 ml Phenol red 1.2 mg or 1.2 ml of 0.1% NaCl 8.0 g KCl 0.4 g or 1.8 ml of 3 M Na2HPO4 45 mg (7H2O) or 1.7 ml of 100 mM KH2PO4 30 mg or 2.2 ml of 100 mM Store in refrigeratior.

TD 1000 ml NaCl 8.0 g KCl 0.38 g or 1.7 ml of 3 M Na2HPO4 0.189 g (7H2O) or 7 ml of 100 mM Tris HCl 3.2 g Tris base 0.59 gpH should be ~7.5. Store in refrigerator.

STE 4000 ml 1000 ml NaCl 150 mM 35.1 g 8.8 g Tris HCl 44 mM 28.1 g 7.0 g Tris Base 6 mM 2.7 g 0.68 g Na2EDTA 1 mM 1.5 g 0.38 g Adjust pH to 7.2.>

Sterilize by autoclaving.

Store in refrigerator.