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=2.0ml
Y
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) T2(add 0.1N HCl) T3(Harvest) Samples
0 15 35 96-11, 96-21, 96-31, 96-41
1 16 36 96-12, 96-22, 96-32, 96-42
2 17 37 72-11, 72-21, 72-31, 72-41
3 18 38 72-12, 72-22, 72-32, 72-42
4 19 39 48-11, 48-21, 48-31, 48-41
5 20 40 48-12, 48-22, 48-32, 48-42
6 21 35 96-11, 96-21, 96-31, 96-41
7 22 36 96-12, 96-22, 96-32, 96-42
8 23 37 72-11, 72-21, 72-31, 72-41
9 24 38 72-12, 72-22, 72-32, 72-42
10 25 39 48-11, 48-21, 48-31, 48-41
11 26 40 48-12, 48-22, 48-32, 48-42
· 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) BSA to add (ul) 0.1N HCl to add (ul)
80 ( A ) 40 stock 960
40 ( B ) 500 ( A ) 500
20 ( C ) 500 ( B ) 500
10 ( D ) 500 ( C ) 500
5 ( E ) 500 ( D ) 500
2.5 ( F ) 500 ( E ) 500