cAMP Accumulation Assay 6/26/2006

Reagent Preparation:

1.  IBMX/DMEM ( 3-isobutyl-1-1-methylxanthine ( Sigma I 5879) ) ---- if need 150ml

Stock Solution: 50mM in 100% EtOH

Working Solution: 1mM in DMEM without serum

X/222.2

0.001= X=33.3 mg

150ml/1000

50mM x 10ˉ³ = 0.0333/222.2 Y=3.0ml

Y

Dissolve 33.3mg IBMX in 3.0ml 100% EtOH, then add to 147ml DMEM without serum.

2.  5 ml CRH-IBMX/ DMEM

Stocking CRH: 300 uM

Working CRH: 100 nM

5 ml x 100nM = Y x 300 x 10³ Y=1.67ul

Add 1.67ul CRH ( 300uM) to 5ml IBMX/DMEM

3.  50 ml Dox-IBMX/DMEM

Dox Stocking: 10ug/ul

Dox Working : 2.0ug/ml

50 ml x 2.0ug/ml = 100ug/ 10ug/ul=10.0ul

Add 10.0 ul of stocking Dox to 50 ml IBMX/DMEM

4.  12 ml CRH/Dox-IBMX/DMEM

Add 4ul CRH (300uM) and 2.4ul Dox(10ug/ul) to 12 ml IBMX/DMEM

5.  0.1 N HCl --- 100ml

12N x Y = 0.1 x 100ml Y=0.83ml

6.  5 ml Dox-IBMX/DMEM Dox series

0.5ug/ml 1.0ug/ml 1.5ug/ml 2.0ug/ml 5ug/ml 10ug/ml

3.3ul of 30uM CRH (1:10 dilute) in 1ml Dox solution =100nM CRH

Induction and Harvest Cells

·  Aspirate media, add 1ml DMEM without serum

·  Aspirate media, add another 1ml DMEM without serum

·  Aspirate media, add 1ml Dox-IBMX/DMEM in Dox wells; add 1ml IBMX/DMEM in Non-Dox wells

·  Incubate 1 hr at RT

·  Aspirate media in wells, add 1ml CRH/Dox-IBMX/DMEM in Dox/CRH wells; add 1ml CRH-IBMX/DMEM in CRH wells, add 1ml Dox-IBMX/DMEM in Dox wells and add 1ml IBMX/DMEM in the rest of wells ( without any labels). incubate 15 min at RT based on the following time points (T1):

T1(add CRH) T2(add 0.1N HCl) Samples

0 15 120-11, 120-21, 120-31, 120-41

1 16 120-12, 120-22, 120-32, 120-42

2 17 96-11, 96-21

3 18 96-12, 96-22

4 19 72-11, 72-21

5 20 72-12, 72-22

6 21 48-11, 48-21

7 22 48-12, 48-22

8 23 24-11, 24-21

9 24 24-12, 24-22

10 25 0.5-1, 1.0-1, 1.5-1

11 26 0.5-2, 1.0-2, 1.5-2

12 27 2.0-1, 5.0-1, 10.0-1

13 28 2.0-2, 5.0-2, 10.0-2

·  After 15min incubation, aspirate media, add 500ul of 0.1N HCl based on T2,

·  Incubate at -4 overnight, next day pipette up and down cell media to lysis cells,

·  Transfer cell lysate to 1.5 ml eppendof tube, keep on ice,

·  Spin at 600g for 5min,

·  Keep supernatant, do protein assay and cAMP assay.

Protein Assay: Use BCA method for protein assay (Range: 20-500ug/ml)

25ul samples standard + 200 ul reagent, check OD at 562nm