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