Microsoft word - 01-camp-prog.doc

Experiment #1: Cyclic AMP and progesterone accumulation in MA-10 cells
Introduction.
Cells will be incubated with buffer only or with a maximally effective concentration of hCG. Cyclic AMP and progesterone (the main steroid product of MA-10 cells) will bemeasured as endpoints at the end of the incubation. The incubation for the cAMPassay will be done in the presence of a phosphodiesterase inhibitor (MIX) to prevent thedegradation of cAMP. At the end of the incubations an aliquot of the medium will besaved for the measurement of progesterone using a commercially available enzyme-linked immunoassay (EIA). The cAMP will be extracted (from the cells and medium)with perchloric acid (PCA). The perchloric acid will then be precipitated from theextracts as potassium perchlorate and cAMP will be measured using a commerciallyavailable enzyme-linked immunoassay (EIA)*.
The kits needed for the cAMP and progesterone EIAs are expensive. A less expensive way to measure them is by using a radioimmunoassay (RIA). This isparticularly true if you raise your own antibodies to cAMP and/or progesterone and ifyou prepare the 125I-cAMP needed as a tracer. For cAMP, the sensitivity of bothassays (either RIA or EIA) is similar and it can be greatly enhanced by acetylating thecAMP standards and samples prior to the assay.
The cAMP and progesterone EIA kits used here are available from Cayman Chemicals.
Original instructions for these kits can be downloaded from www.caymanchem.com Expected Results
Progesterone and cAMP levels should be low in the MA-10 cells transfected with the empty vector (EV) as well as in the hLHR-wt transfected cells incubated with buffer only.
These levels should be increased by hCG stimulation. Progesterone and cAMP levelsshould be high in the MA-10 cells expressing hLHR-L457R and incubated with bufferonly. They should be stimulated little (if any) by addition of hCG.
Basal and hCG-stimulated progesterone and cAMP levels in the cells transfected with the hLHR-wt and a control siRNA should be similar to those of cells transfectedwith hLHR-wt only (see above). The siRNA against Gαs should decrease hCG-stimulated cAMP accumulation by ~50%. It may or may not decrease hCG-stimulatedprogesterone accumulation. The siRNA against Gα11 should not affect cAMP orprogesterone accumulation.
Additional References:
Quintana J, Wang H, Ascoli M 1993 The regulation of the binding affinity of theluteinizing hormone/choriogonadotropin receptor by sodium ions is mediated by ahighly conserved aspartate located in the second transmembrane domain of Gprotein-coupled receptors. Mol. Endocrinol. 7: 767-775 Harper JF, Brooker G 1975 Femtomole sensitive radioimmunoassay for cyclicAMP and cyclic GMP after 2'O acetylation by acetic anhydride in aqueous solution.
J. Cyclic Nucl. Res. 1: 207-218 Brooker G, Harper JF, Terasaki WL, Moylan RD 1979 Radioimmunoassay of cyclicAMP and cyclic GMP. Advances in Cyclic Nucleotide Res. 10: 1-33 Stimulation of cells and preparation of extracts for progesterone and cAMP
1. Assay medium: DMEM/F12 with 20 mM Hepes, 1 mg/ml BSA and 50 µg/ml gentamicin, pH 7.5. Store sterile at 4ºC. Warm up before using.
2. Assay medium with 1 mM MIX. Make on the day of the assay by dissolving MIX into the assay medium at a concentration of 0.22 mg/ml. Keep cold.
3. 0G/BSA. 20 mM Hepes, 0.1 M NaCl, pH 7.4 with 1 mg/ml BSA.
5. 2N PCA. Dilute 86 ml concentrated (70%) PCA to 500 ml with water. Add 360 mg theophylline. Store in the refrigerator. Theophylline is a phosphodiesteraseinhibitor.
6. 0.72 M KOH/0.6 M KHCO3. For 250 ml of solution: 10.1 g KOH and 15.0 g KHCO3.
7. MA-10 cells. Each group will have 12 wells of MA-10 cells.
For groups A and B cells will be pre-transfected with an empty vector (label these 1-4),the hLHR-wt (label these 5-8) or with hLHR-L457R (label these 9-12). Group A will usethese cells to measure progesterone and Group B will measure cAMP.
For groups C and D cells will be pre-transfected with the hLHR-wt and a control siRNA(label these 1-4), the hLHR-wt and the siRNA against Gαs (label these 5-8) or with thehLHR-wt and the siRNA against Gα11 (label these 9-12). Group C will use these cells tomeasure progesterone and Group D will measure cAMP.
1. Wash the cells twice with 1-2 ml aliquots of warm Assay medium.
After the last wash groups A and C will add 1 ml of warm Assay medium to each well.
These cells will be used to measure progesterone.
After the last wash groups B and D will add 1 ml of warm Assay medium with 1 mM MIXto each well. These cells will be used to measure cAMP.
2. Place the wells in the incubator for 15 min and then make the following additions: Wells 1, 2, 5, 6, 9 and 10: 50 µl of OG/BSAWells 3, 4, 7, 8, 11 and 12: 50 µl of 2 µg/ml hCG (final concentration = 100 ng/ml) 3. Mix the wells gently and put them back in the incubator for 30 min (groups B and D) or 4 hours (groups A and C). At the end of this incubation groups A and C shouldproceed with step 4. Groups B and D should skip step 4 and proceed with step 5.
4. Groups A and C should mix the wells gently and save 500 µl of medium from each well in cold 12 x 75 mm tubes. Centrifuge the tubes at full speed in the table topcentrifuge at 4C for 10 min and collect the supernatants into new tubes. Storefrozen until Wednesday for the progesterone EIA.
5. The next steps apply only to groups B and D: Place the wells on ice and add 1 ml of 2N PCA to each well. Swirl to mix and incubate on ice for 30-60 min.
6. Collect the PCA extracts (while leaving the cells behind) and transfer them to cold 12 x 75 mm tubes and centrifuge these tubes at full speed in the table top centrifuge at4C for 10 min.
7. Save 1 ml of the supernatants (from step 6 above) into new 12 x 75 tubes and then add 0.5 ml of 0.72 M KOH/0.6 M KHCO3 . Vortex and centrifuge as in step 6 to remove the salt precipitate. Save the supernatants into new tubes. These will beused for the cAMP EIA as outlined below.
Setting up the cAMP EIA.
Cyclic AMP EIA (modified from Cayman protocol) 4 N KOHFor 100 ml: Dissolve 22.44 g KOH in water.
Store at room temperature.
Acetic anhydrideProvided with the kit.
Store at room temperature.
cAMP standardPrepare a 7.5 µM standard solution by reconstituting the vial provided with the kitwith 1 ml EIA buffer.
Dilute 15 µl of this standard + 11.23 ml EIA buffer to give a 10 pmol/ml standard Prepare EIA and Wash buffers as recommended by manufacturer and store at 4C Reconstitute the cAMP-acetylcholinesterase tracer (vial #2 in kit) with EIA buffer asrecommended in the instructions (the amount of buffer to use depends on whetherwe buy kits for single plates or a kit for 5 plates) and store at 4ºC. Check theinstructions that come with the kit but this is usually 6 ml of buffer for the singleplate kit and 30 ml of buffer for the 5 plate kit.
Reconstitute the cAMP antiserum (vial #1 in kit) with EIA buffer as recommendedin the instructions (the amount of buffer to use depends on whether we buy kits forsingle plates or a kit for 5 plates) and store at 4ºC. Check the instructions thatcome with the kit but this is usually 6 ml of buffer for the single plate kit and 30 mlof buffer for the 5 plate kit.
Samples 1-6: 100 µl of sample + 400 µl of EIA buffer (5 X dilution)Samples 7-12: 5 µl of sample + 495 µl of EIA buffer (100 X dilution) Samples 1,2,5,6 and 9,10: 100 µl of sample + 400 µl of EIA buffer (5 X dilution)Samples 3,4,7,8 and 11,12: 5 µl of sample + 495 µl of EIA buffer (100 X dilution) Prepare the cAMP standards by diluting the 10 nM (10 pmol/ml) cAMP standard asfollows: S1 = Use 10 pmol/ml solution made aboveS2 = 500 µl of S1 standard + 500 µl of EIA bufferS3 = 500 µl of S2 standard + 500 µl EIA bufferS4 = 500 µl of S3standard + 500 µl EIA bufferS5 = 500 µl of S4 standard + 500 µl EIA bufferS6 = 500 µl of S5 standard + 500 µl EIA bufferS7 = 500 µl of S6 standard + 500 µl EIA bufferS8 = 500 µl of S7 standard + 500 µl EIA buffer Acetylate** all the diluted samples and standards (be sure that each tube to be
acetylated has 500 µl of sample or standard)
Add 100 µl of 4N KOH to each tube
Add 25 µl of acetic anhydride to each tube and vortex immediately for 15 sec
Add 25 µl of 4N KOH to each tube
Incubate for 30 min at room temperature
** This is a tricky procedure and it will be done either by the instructor or the
teaching assistants.
Group B. Dilute the acetylated samples as follows: C10, D10 100 µl of acetylated sample # 10 400 µl G10, H10 50 µl of acetylated sample # 10 C11, D11 10 µl of acetylated sample # 10 100 µl of acetylated sample # 11 400 µl G11, H11 100 µl of acetylated sample # 12 400 µl C12, D12 50 µl of acetylated sample # 12 G12, H12 10 µl of acetylated sample # 12 Group D. Dilute the acetylated samples as follows: C10, D10 50 µl of acetylated sample # 10 G10, H10 25 µl of acetylated sample # 10 100 µl of acetylated sample # 11 400 µl G11, H11 100 µl of acetylated sample # 12 400 µl C12, D12 50 µl of acetylated sample # 12 G12, H12 10 µl of acetylated sample # 12 Take out the 96 well plate and rinse the wells once with 200 µl of wash buffer.
Discard the contents of the plate by inverting and tapping firmly on a stack ofabsorbent paper.
Make additions to numbered wells of the EIA plates as follows Cover the plate with the plastic cover provided and incubate for 18 hours at 4ºC STOP HERE AND CONTINUE WITH STEP 8 TOMORROW
Dilute the vial with Ellman’s Reagent (vial # 8 in kit) with water as recommended inthe instructions (the amount of water to use depends on whether we buy kits forsingle plates or a kit for 5 plates). Check the instructions that come with the kit butthis is usually 20 ml of water for the single plate kit (for 1 vial of Ellman’s reagent)and 60 ml of buffer for the 5 plate kit (for 3 vials of Ellman’s reagent).
Discard the contents of the plate by inverting and tapping firmly on a stack ofabsorbent paper. Add 200 µl of wash buffer to each well. Discard the contents ofthe plate as above and repeat four more times to give a total of 5 washes.
10 Add 200 µl of Ellman’s reagent to each well. Cover and shake in the dark in an orbital shaker for 60-90 min or until Bo (wells 3 and 4) read ~ 0.4.
Calculations of the amount of cAMP generated will be done using a spreadsheetprovided. The EIA may have to be repeated if the samples are out of the standardcurve.
All data will be corrected for cell density. The teaching assistants will count thecells on the day of the experiment.
Progesterone EIA (modified from Cayman protocol) 1. Progesterone standard: Equilibrate a yellow pipet tip with 200 µl of ethanol by repeatedly filling it up and down with ethanol. Use this pipet tip to measure 100 µl ofthe progesterone standard supplied into a clean test tube and dilute it with 900 µl ofwater to give a 10 ng/ml standard. Store at 4ºC.
2. Assay medium: DMEM/F12 with 20 mM Hepes, 1 mg/ml BSA and 50 µg/ml gentamicin, pH 7.5. Sterile. Warm up before using 3. Prepare the EIA and Wash buffers as recommended by manufacturer and store at 4. Reconstitute the progesterone-acetylcholinesterase tracer (vial #2 in kit) with 6 ml of EIA buffer (for the single plate kits) or 30 ml of EIA buffer (for the 5 plate kits) andstore at 4C 5. Reconstitute the progesterone antiserum (vial #1 in kit) with 6 ml of EIA buffer (for the single plate kits) or 30 ml EIA buffer (for the 5 plate kits) and store at 4C Prepare the progesterone standards by diluting the 10 ng/ml progesterone stock asfollows (vortex well before proceeding with dilutions): Std 8 = 1000 pg/ml standard = 100 µl of 10 ng/ml progesterone stock + 900 µl ofEIA buffer (50 pg/50 µl)Std 7 = 500 pg/ml standard = 500 µl of 1000 pg/ml standard + 500 µl of EIA buffer(25 pg/50 µl)Std 6 = 250 pg/ml standard = 500 µl of 500 pg/ml standard + 500 µl of EIA buffer(12.5 pg/50 µl)Std 5 = 125 pg/ml standard = 500 µl of 250 pg/ml standard + 500 µl of EIA buffer(6.25 pg/50 µl)Std 4 = 62.5 pg/ml standard = 500 µl of 125 pg/ml standard + 500 µl of EIA buffer(3.13 pg/50 µl) Std 3 = 31.25 pg/ml standard = 500 µl of 62.5 pg/ml standard + 500 µl of EIA buffer(1.56 pg/50 µl)Std 2 =15.63 pg/ml standard = 500 µl of 31.25 pg/ml standard + 500 µl of EIAbuffer (0.78 pg/50 µl)Std 1 = 7.8 pg/ml standard = 500 µl of 31.25 pg/ml standard + 500 µl of EIA buffer(0.39 pg/50 µl) Samples 1-12: 20 µl of sample + 980 µl of EIA buffer (50 X dilution) Group A: dilute your samples more as follows Group C: dilute your samples more as follows Take out the 96 well plate and rinse the wells once with 200 µl of wash buffer.
Discard the contents of the plate by inverting and tapping firmly on a stack ofabsorbent paper.
Make additions to numbered wells of the EIA plates as follows A4-H12 Samples 50 µl diluted samples 50 µl of prog tracer Cover the plate with the plastic cover provided and incubate for 1 hour at roomtemperature on an orbital shaker Dilute the vial with Ellman’s Reagent (vial # 8) with 20 ml of water (for one plate kit:1 vial of Ellman’s reagent) or 60 ml of water (for 5 plate kit: 3 vials of Ellman’sreagent) and keep it covered with aluminum foil Discard the contents of the plate by inverting and tapping firmly on a stack ofabsorbent paper. Add 200 µl of wash buffer to each well. Discard the contents ofthe plate as above and repeat four more times to give a total of 5 washes.
Add 200 µl of Ellman’s reagent to each well. Cover and shake in the dark in anorbital shaker for 60-90 min or until Bo (wells 3 and 4) read ~ 0.4.
14 The teaching assistants will determine the number of cells present in each well and all calculations of the amount of progesterone generated will be done using aspreadsheet provided. The EIA may have to be repeated if the samples are out ofthe standard curve.

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