Materials Reagents

2.1.1. Perfusion of the Mouse Pancreas In Situ

1. Krebs-Ringer bicarbonate buffer (KRB): 4.4 mM KCl, 2.1 mM CaCl2, 1.5 mM KH2PO4, 1.2 mM MgSO4, 29 mM NaHCO3, and 116 mM NaCl prepared on the day prior to perfusion.

2. Dextran-70: (cat. no. 17-0280-02, Amersham). Prepare a 3% (w/v) solution in KRB to form KRB-dextran on the day prior to perfusion.

3. Nembutal sodium solution: 50 mg/mL (cat. no. NDC-0074-3778-04, Abbott) diluted 1:10 in 0.9% NaCl. Store up to 1 mo at 48C.

5. Arginine-HCl (cat. no. A6757, Sigma Chemical Co., St. Louis, MO).

6. Bovine serum albumin (BSA): Fatty acid free (cat. no. A-6003, Sigma).

7. Sodium chloride: Sterile, 0.9% (w/v) NaCl (cat. no. NDC-0074-4888-10, Abbott Laboratories, North Chicago, IL).

2.1.2. Insulin Immunoassay of Samples Acquired During Perfusion of the Mouse Pancreas In Situ

1. Phosphate buffer (pH 7.4): 0.063 M Na2HPO4, 0.013 M C10H14O8Na2-2H2O, and 0.003 M NaN3 (see Note 1). Store at 4°C for up to 1 mo.

2. Bovine serum albumin (BSA): see Subheading 2.1.1., item 6 (see Note 2).

3. Radioimmunoassay (RIA) buffer: 0.5% w/v BSA in phosphate buffer. Store at 4°C for up to 1 wk.

4. Rat insulin reference standard: Prepare by dissolving the contents of one vial of rat insulin (cat. no. 8013, Linco Research, Inc., St. Charles, MO) in 6.25 mL RIA buffer to form 16 ng insulin/mL (standard A). Prepare additional solutions of insulin by diluting standard A with RIA buffer as follows: (1) 1.55 mL A + 1.55 mL RIA buffer = 8 ng/mL, (2) 0.8 mL A + 2.4 mL RIA buffer = 4 ng/mL, (3) 0.4 mL A + 2.8 mL RIA buffer = 2 ng/mL, (4) 0.2 mL A + 3.0 mL RIA buffer = 1 ng/mL, (5) 0.1 mL A + 3.1 mL RIA buffer = 0.5 ng/mL, and (6) 0.05 mL A + 3.15 mL RIA buffer = 0.25 ng/mL. Store 0.25-mL aliquots of each standard solution (12 for each concentration) at —70°C for up to 1 yr.

6. 125I-Insulin: Use according to manufacturer's instructions (see Note 3) (cat. no. TIN2, Diagnostic Products Corporation, Los Angeles, CA).

2.1.3. Intraperitoneal Glucose Tolerance Testing in the Mouse

1. Phosphate-buffered saline (PBS): 10 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl. Store at 4°C.

2. Glucose solution: Prepare 0.1 g/mL dextrose in PBS as needed.

2.1.4. Insulin Immunoassay of Plasma Acquired During Glucose Tolerance Testing in Wild-Type or Transgenic Mice

1. Phosphate buffer (pH 7.4): see Subheading 2.1.2., item 1.

2. Triton™ X-100 (cat. no. 161-0407, Bio-Rad Laboratories, Hercules, CA): Prepare a 10% (v/v) solution in phosphate buffer and store at 4°C for up to 6 mo (see Note 4).

3. Aprotinin (cat. no. 616398, Calbiochem-Novabiochem, La Jolla, CA): Prepare a 100,000-K.I.U./mL solution by dissolving 500 KU (500,000 K.I.U.) in 5 mL of phosphate buffer. Store 0.25-mL aliquots at — 70°C for up to 1 yr.

4. RIA buffer: Prepare by adding 1 mL 10% (v/v) Triton™ X-100 and 0.2 mL (20,000 kIU) aprotinin to 98.8 mL phosphate buffer (see Notes 4 and 5). Store at 4°C for up to 1 wk.

5. Rat insulin standard: Prepare by dissolving the contents of one vial of rat insulin (cat. no. 8013, Linco Research, Inc.) in 10 mL RIA buffer to form 10 ng insulin/mL. Store 0.2-mL aliquots at -70°C for up to 1 yr. As needed, dilute 0.1 mL of the 10 ng/mL with 4.9 mL RIA buffer to form 200 pg insulin/mL. Prepare additional solutions of insulin (in RIA buffer) of 100, 50, 20, 10, and 5 pg/mL.

6. Samples: Mouse plasma, stored at -70°C until assayed. As needed, transfer entire sample to a 1.5-mL conical tube (Eppendorf). Record the sample volume. Add enough RIA buffer to form 0.3 mL diluted plasma. Record the dilution factor.

7. Normal guinea pig serum (NPGS): Prepare by dissolving the contents of one vial of lyophilized normal guinea pig serum (cat. no. 7020-25, Linco Research, Inc.) in water as recommended. Store 0.5-mL aliquots at -70°C for up to 2 yr. As needed, dilute 1: 50 with RIA buffer to form NGPS buffer (see Note 6).

8. Primary antibody (insulin antibody): Prepare by dissolving the contents of one vial of guinea pig anti-rat insulin serum (cat. no. 1013, Linco Research, Inc.) in 10 mL NGPS buffer. Store 0.5 mL aliquots at -70°C for up to 2 yr. As needed, dilute the primary antibody 1 : 70 in NGPS buffer.

9. Radioactive insulin: Prepare by dissolving the contents of one vial of 125I-human insulin (cat. no. 9011, Linco Research, Inc.) in 10 mL of RIA buffer. Store 1 mL aliquots at -70°C for up to 2 mo. As needed, dilute the 125I-insulin to 5-6000 cpm/0.1 mL with RIA buffer (see Note 7).

10. Secondary antibody (antibody to primary antibody): Prepare by dissolving the contents of one vial of goat anti-guinea pig gamma globulin serum (cat. no. 5020-20, Linco Research, Inc.) in water as recommended. Store 0.5-mL aliquots at -70°C for up to 2 yr. As needed, dilute the secondary antibody 1:30 with phosphate buffer.

11. Separation buffer: Prepare by dissolving 2.5 g BSA (cat. no. 60069, ICN Pharmaceuticals, Inc.) and 3.0 g polyethylene glycol 8000 (PEG) (cat. no. BP233-1, Fisher Scientific) in 100 mL phosphate buffer. Stand in ice water until used.

2.1.5. Human Insulin Immunoassay of Plasma Acquired During Glucose Tolerance Testing in Mice Bearing Xenografts of Either Human Islets or Cells Engineered to Secrete Human Insulin

2. Human insulin standard: Prepare by dissolving the contents of one vial of human insulin (cat. no. 8014, Linco Research, Inc.) in 10 mL of RIA buffer to form 10 ng insulin/mL. Store 0.2-mL (2-ng) aliquots at -70°C for up to 1 yr. As needed, add in 1.8 mL of RIA buffer to a 0.2-mL (2-ng) aliquot of human insulin to form 1000 pg/mL. Prepare additional solutions of human insulin (in RIA buffer) of 500, 200, 100, 50, 20, 10, and 5 pg/mL.

3. Samples: Mouse plasma, stored at -70°C until assayed. As needed, transfer the entire sample to a 1.5-mL conical tube (Eppendorf). Record the sample volume. Add in enough RIA buffer to form 0.475 mL diluted plasma. Record the dilution factor. Reserve one-half of each diluted sample for use in the mouse plasma insulin RIA, which measures total plasma insulin (human insulin secreted by the grafted cells plus mouse insulin secreted by the host).

Fig. 2. Diagrammatic representation of the perfusion chamber.

5. Primary antibody (human insulin antibody): Prepare by dissolving the contents of one vial of guinea pig anti-human insulin serum (cat. no. 1014, Linco Research, Inc.) in 10 mL of NGPS buffer. Store 0.5-mL aliquots at -70°C for up to 2 yr. As needed, dilute the primary antibody 1:30 with NGPS-buffer.

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Responses

  • uta
    How to prepare KRB buffer?
    7 years ago

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