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Yu-li Wang's Laboratory: Lab Protocols

Solutions for Tissue/Cell Digestion

Trypsin (13 ug/ml) in Saline G

Materials

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 -20oC.

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 -70oC.

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 -70oC 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 -70oC 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 ml
of 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 g
pH 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.


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