hGH transfection reagents & protocols

The pXGH5 plasmid is available from Nichols Institute Diagnostics as part of the ‘Allegro HGH Transient Gene Expression System’ which is a high sensitivity radiometric assay kit for hGH. Nichols telephone in USA is 800 642-4657 or 714 728-4000. In Germany, the number is (06032) 91060. Attached are protocols for transfection of PC12 cells.

WILSON TRANSFECTION PROTOCOL

November 1, l996

PC - 12 cells

(See Wilson et al. , 1995, Anal. Biochem. 226:212-220 for development of method)

1. 2.4x 106 P C - 12 cells were plated in 35.0 mm (1.2 x 10 6 cells in 22.6 mm) tissue culture wells about 18 hours before transfection using the appropriate growth medium . Cells were then incubated at 37 C in a humidified, 10% CO2 environment.

2. Cells were fed with 1.8 ml/ well of DMEM media + 0% FCS + Pen-Strep and Gentamicin (o CAF) + 0 Fungizone for 2 hours before transfection. (0.9 ml/ 22.6 mm well)

3. 0.200 ml of a calcium phosphate - DNA suspension was prepared for each 35.0 mm well of the cells as follows (Half this amount was prepared for 22.6 mm wells). Use only sterile conical polypropylene tubes.

a. Prepare 0.100 ml/well of solution containing 100 ug/ml DNA in 250 mM CaCl2 for each well and then dilute to 0.200 ml/well with 2 x PiBS.

b. To the above solutions, add an equal volume of 280 mM NaCl + 1.5 mM Phosphate + 40 mM PIPES (2xPiBS) solution while vortexing. The resulting solution should consist of 50 ug/ml DNA in 125 mM CaCl2 + 140 mM NaCl + O.75 mM Na Phosphate + 20 mM PIPES. Mix one additional second and then let stand for 30-40 minutes. 200 ul of this final solution was added dropwise to each 35.0 mm or 22.6 mm (100 ul) well. Cells were placed in a 10% CO2incubator for 4.0 hours before they were glycerol shocked.

c. Cells were glycerol shocked by adding 1.0 ml per 35.0 mm well or 0.5 ml per 22.6 mm well of 12.5% glycerol in serum-free DMEM media containing glutamine but no antibiotics. This media was pre-equilibrated at 10% CO2 for several hours. Wait exactly 3 minutes and add 2.0 ml or 1.0 ml of culture medium to the glycerol solution. Aspirate and wash the cultures once with 2.0 (1.0)ml media per well. Aspirate again and then add 2.0 (1.0) ml/well culture media containing serum, and antibiotics. Finally, return cultures to the 10% CO2 incubator. Experiments were performed two or three days after the glycerol shock..

PC-12 CELL SECRETION PROTOCOL

FOR TRANSFECTED CELLS

January 10, 1997

PC12 secretion transfected

A. Cells were loaded with 500 ul/ 22.6 mm well of 20 ml media containing 30 ul Na Ascorbate (50 mg/ml) and 30 ul of (3H)norepinephrine (1m Ci/ml) the night before the experiment. Cells were labelled overnight.

B. Medium was removed and cells were rinsed 2x’s with 1.0 ml/well of CaPSS containing 5.6 mM Glucose, 0.5 mM sodium ascorbate, and 1 mg/ml BSA.

C. Cells were then incubated for an additional 2 hours in 0.8 ml fresh culture medium/ 22.6 mm well.

D. Medium was replaced with 1.0 ml CaPSS + G + NaAscorbate + 1 mg/ml BSA 20 to 45 minutes before starting the experiment.

E. Experiment commenced with the addition of 0.6 ml/ 22.6 mm well CaPSS + G+ NaAs +1 mg/ml BSA containing 0 or 30 uM Ionomycin or 100 mM KPSS. The test solutions were removed after 15 minutes.

F. The amount of (3H) norepinephrine released into the test solution was determined radiometrically after the test solutions were centrifuged by sampling 50 ul from each test solution. Growth Hormone levels were determined by sampling 400 ul of the test solution and assaying for Growth Hormone using the Nichols Institute Chemiluminenence Growth Hormone detection system (The radiometric assay in ‘Allegro’ kit from Nichols is also suitable).

G. The amount of (3H) norephinephrine remaining in the cells was measured by lysing the cells with 0.6ml lysis buffer (10 mM Hepes + 0.2 mM diNa EDTA) containing 0.1% Triton X-100, and scraping each 22.6 mm well. 50 ul was sampled from each well and the radioactivity was measured . Growth Hormone was determined by sampling 100ul and assayed as above. Since the Triton alters the hGH assay, run standards both with and without TritonX100.