cAMP Accumulation Assay
Reagent Preparation:
1. IBMX/DMEM ( 3-isobutyl-1-1-methylxanthine ( Sigma I 5879) ) ---- if need
100ml
Stock Solution: 50mM in 100% EtOH
Working Solution: 1mM in DMEM without serum
X/222.2
0.001=
X=22.2 mg
100ml/1000
50mM x 10ˉ³ = 0.0222/222.2
Y
Y=2.0ml
Dissolve 22.2mg IBMX in 2.0ml 100% EtOH, then add to 98ml DMEM without serum.
2. 15 ml CRH-IBMX/ DMEM
Stocking CRH: 300 uM
Working CRH: 100 nM
12 ml x 100nM = Y x 300 x 10³
Y=4ul
Add 4ul CRH ( 300uM) to 12ml IBMX/DMEM
3. 40 ml Dox-IBMX/DMEM
Dox Stocking: 10ug/ul
Dox Working : 1.5ug/ml
40 ml x 1.5ug/ml = 60ug/ 10ug/ul=6.0ul
Add 6.0 ul of stocking Dox to 40 ml IBMX/DMEM
4. 12 ml CRH/Dox-IBMX/DMEM
Add 4ul CRH (300uM) and 1.8 ul Dox(10ug/ul) to 12 ml IBMX/DMEM
5. 0.1 N HCl --- 100ml
12N x Y = 0.1 x 100ml
Y=0.83ml
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 CRH 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)
0
1
2
3
4
5
T2(add 0.1N HCl) T3(Harvest)
15
35
16
36
17
37
18
38
19
39
20
40
Samples
96-11, 96-21, 96-31, 96-41
96-12, 96-22, 96-32, 96-42
72-11, 72-21, 72-31, 72-41
72-12, 72-22, 72-32, 72-42
48-11, 48-21, 48-31, 48-41
48-12, 48-22, 48-32, 48-42
6
7
8
9
10
11
21
22
23
24
25
26
96-11, 96-21, 96-31, 96-41
96-12, 96-22, 96-32, 96-42
72-11, 72-21, 72-31, 72-41
72-12, 72-22, 72-32, 72-42
48-11, 48-21, 48-31, 48-41
48-12, 48-22, 48-32, 48-42





35
36
37
38
39
40
Clone
12-0-2
Clone
2-4-3
After 15min incubation, aspirate media, add 500ul of 0.1N HCl based on T2,
After 20min, 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, then store at -20 for next day’s cAMP assay.
Protein Assay: Due to low protein concentration, we need use Bio-Rad method for
protein assay (Range: 0-80ug/ml)
160 ul samples ( 1:5 or 1:8 dilution) or standard + 40 ul reagent ( 1:5 dilution) , check OD
at 595nm
Stock BSA: 2000ug/ml
Final BSA Con(ug/ml)
80 ( A )
40 ( B )
20 ( C )
10 ( D )
5 (E)
2.5 ( F )
BSA to add (ul)
40 stock
500 ( A )
500 ( B )
500 ( C )
500 ( D )
500 ( E )
0.1N HCl to add (ul)
960
500
500
500
500
500