GE Healthcare
HitHunterTM cAMP XS FL Assay
FLUORESCENT DETECTION
Product Booklet
Codes:
DX90008102 (Number of wells 800, recommended format 384 well)
DX90008103 (Number of wells 10 000, recommended format 384 well)
DX90008104 (Number of wells 40 000, recommended format 384 well)
PAGE FINDER
LEGAL SECTION
Page 3
INTENDED USE
Page 4
TECHNOLOGY PRINCIPLE
Page 4
STORAGE CONDITIONS
Page 4
MATERIALS PROVIDED
Page 4
MATERIALS NOT PROVIDED
Page 5
GUIDELINES FOR USE
Page 5
TIPS FOR OPTIMAL PERFORMANCE
Page 5
REAGENT PREPARATION
Page 5
PROCEDURE
Page 6
REPRESENTATIVE DATA
Page 7
QUICK START GUIDE
Page 9
NOTES
LEGAL SECTION
GE, imagination at work and GE Monogram are trademarks of General Electric Company.
DiscoveRx name, logo, and HitHunter are trademarks of DiscoveRx Corp.
Galacton Star (registered in the U.S.) and Emerald II are trademarks of Applied Biosystems.
Grants to the purchaser of this product an end-user license under United States and foreign patents
owned or licensed exclusively to DiscoveRx (including US patent numbers, 4708929, 4956274, 5244785,
5444161, 5604091, 5643734 and equivalent foreign patents) and non-exclusively licensed (including US
patent numbers 5851771, 5538847, 5326882, 5145772, 4978614, and 4931569 and equivalent foreign
patents) to use this product solely in the fields of drug discovery and regulatory qualification of
therapeutic drugs and not for any other uses, including but not limited to, other research uses, or use in
human clinical, animal, or food diagnostics.
© 2007 DiscoveRx Corporation - All rights reserved
© 2006-2007 General Electric Company – All rights reserved
Previously published 2006
All goods and services are sold subject to the terms and conditions of sale of the company within GE
Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact
your local GE representative for the most current information.
www.gehealthcare.com/lifesciences
GE Healthcare UK Limited
Amersham Place, Little Chalfont
Buckinghamshire, HP7 9NA
UK
- 10 10
-3 3
INTENDED USE
HitHunterTM cAMP XS FL is used to monitor cAMP in cell lysates produced from whole cells induced in a
microplate format.
TECHNOLOGY PRINCIPLE
Enzyme fragment complementation (EFC) technology uses two fragments of E. coli E-galactosidase
(E-gal): a large protein fragment (enzyme acceptor, EA) and a small peptide fragment (enzyme donor, ED).
Separately, these fragments are inactive, but in solution they rapidly complement (recombine) to form
active ź-gal enzyme, which can hydrolyze substrate to produce a Fluorescent signal.
In this assay, cAMP from cell lysates and ED-labeled cAMP (ED-cAMP) compete for antibody binding sites.
Unbound ED-cAMP is free to complement EA to form active enzyme, which subsequently produces a
fluorescent signal. The amount of signal produced is proportional to the amount of cAMP in the cell lysate.
STORAGE CONDITIONS
Upon arrival, store reagents at ” -20°C. The kit is stable until the expiry date indicated on the outer kit box
label. Thaw reagents at room temperature before use, and after thawing, store reagents for up to 1 month
at 2–8°C. For longer term storage, aliquots of all the components may be re-frozen at ” -20°C. The
reagents can be thawed and frozen for a TOTAL of 3 times without loss in performance.
MATERIALS PROVIDED
The HitHunter cAMP XS FL Assay kit contains the following components:
1
2
3
4
5
6a
6b
7
Product Code
Test points in 96-well format
Test points in 384-well format
Kit Components
cAMP XS FL Lysis Buffer1
cAMP XS FL EA Reagent
cAMP XS FL ED Reagent
cAMP XS FL Antibody Reagent
cAMP XS FL Standard (250 µM)
cAMP XS FL Substrate
cAMP XS FL Dilution Buffer
Stop Solution
DX90008102
DX90008103
DX90008104
400
5000
20 000
800
10 000
40 000
Volume in each bottle (mL)
4
50
200
16
200
800
8
100
400
4
50
200
1
13
52
1.6
20
80
6.4
80
320
8
80
320
Note: 1cAMP Lysis Buffer (pH 6.9) contains 10 mM phosphate, 10 mM NaCl, lysing agent, and
other proprietary components.
Reagents from cAMP XS FL kit are not exchangeable with other HitHunter cAMP kits.
-4 4
9
FIGURE 2: Cell response data for HitHunter cAMP XS FL run in a 384-well plate.
MATERIALS NOT PROVIDED
The following additional materials are required:
Equipment
x 96 well or 384 well black Microplates
x Pipettes and tips
x Microtiter plate Fluorometer or CCD
imager
x Filters (Ex: 530±20 or 560±20, EM: >590)
c A M P XS F L F or s k ol i n I n d u c t i on
C H O K 1 20 000 c e l l s / we l l
8000
RFU
6000
CHO K1: 20K/well
EC50 3.4 PM
S:B 24.4
x Serum free culture media with or without
phenol red
x IBMX (Sigma #I5879 or #I7018);
x PBS (Sigma# D8537) or similar
GUIDELINES FOR USE
HitHunter cAMP XS FL is a homogenous, 3 step assay. Cells are plated and induced in PBS or media, and
cell induction is followed by 3 reagent additions and 2 incubation steps. Please read fluorescence at
530 nm excitation ± 25 nm, 610 nm emission ± 20 nm.
4000
TIPS FOR OPTIMAL PERFORMANCE
2000
0
10 -8
Materials
x
10 -7
10 -6
10 -5
F or s k ol i n [ M ]
10 -4
10 -3
x
x
x
x
x
Titer cell number to establish the optimum cell concentration for each cell line. For example, if cells
will be assayed in suspension in a 384-well plate format, an initial cell number titration may include
5000, 10 000, and 20 000 cells per well.
Recommended controls:
Basal level: measure cAMP in untreated cells
EFC background: substitute PBS for ED reagent to obtain background EFC signal.
Protect the reagents from light when possible and cover the microplate with foil or a black plate
during all incubations.
Addition of chelating agents, reducing agents, some detergents or changes in pH may adversely
affect assay performance.
The plate may be incubated overnight and the signal may be measured the next day. The actual
signal characteristics over time are affected by lab conditions such as temperature and the user
should establish an optimal read time.
Protocols are provided in 384 well formats, for 96-well format, multiply all volumes by a factor of 2.
REAGENT PREPARATION
Thaw and equilibrate reagents completely to ambient room temperature before use.
cAMP XS FL EA Reagent - Ready to use.
cAMP XS FL ED Reagent - Ready to use
cAMP XS FL Stop Reagent - Ready to use
cAMP XS FL Antibody Reagent – Mix equal part Ab and lysis buffer to make Ab / Lysis working reagent
-8 8
-5 5
CAMP XS FL ANTIBODY / LYSIS /FL SUBSTRATE WORKING SOLUTION –
aspirate growth medium from the wells. Add 10 µL of serum free media, and then follow cell-based assay
procedure from step 2.
1.Add 1 part FL Substrate to 4 parts FL Dilution Buffer to make FL Working Reagent and mix by inversion.
2.Add 1 part Antibody / Lysis working reagent to 1 part FL Working Reagent and mix by inversion
Assay Procedure:
Note: The Ab / Lysis /FL Substrate Working Solution may be used at room temperature for up to 24
hours.
384-well Plate
cAMP XS FL Protocol: Cell Based
Step 1
10 µL of cells diluted in PBS or serum free media
PROCEDURE:
Step 2
5 µL compound
Standard Curve Protocol
Step 3
Induction Incubation
Step 4
20 µL Antibody/ Lysis/ susbtrate Working Solution
Step 5
10 µL ED Reagent
Step 6
Incubate 1 hour at room temperature
Step 7
20 µL EA Reagent
Step 8
Incubate 4 hour at room temperature, then read fluorescent signal
530nm excitation ± 25nm
610nm emission ± 20nm
CAMP STANDARD – Into polypropylene tubes, dilute the cAMP Standard (2.5 x 10–4 M): 1
part to 8 parts PBS;
this dilution is the highest standard concentration at 6.4 x 10-6 M in the final 65 µL assay volume.
Make 9 additional 3-fold serial dilutions, with the last dilution yielding the lowest standard concentration
of 3.3 x 10-10 M in the final 65 µL assay volume. Use PBS for the zero standard.
ASSAY PROCEDURE
384-well Plate
cAMP XS FL Protocol: Standard Curve
Step 1
15 µL of diluted cAMP standard
Step 2
20 µL Antibody/ Lysis/ Susbtrate Working Solution
Step 3
10 µL ED Reagent
Step 4
Incubate 1 hour at room temperature
Step 5
20 µL EA Reagent
Step 6
Incubate 4 hour at room temperature, then read fluorescent signal
530nm excitation ± 25nm
610nm emission ± 20nm
Notes: The plate may be incubated overnight and the signal may be measured the next day. In this case
add 7 µL Stop Solution after the 4 hour incubation to stabilize the signal. Conditions for agonist and
antagonist induction must be established for each cell line.
REPRESENTATIVE DATA
Figure 1: Standard Curve for HitHunter cAMP XS FL run in a 384-well plate.
c A M P XS F L S ta n d a r d C u r v e
NOTE: The plate may be incubated overnight and the signal may be measured the next day.
In this case add 7 ƄL Stop Solution after the 4 hour incubation to stabilize the signal.
10000
8000
CELL PREPARATION FOR CELLS IN SUSPENSION - Harvest and resuspend cells in PBS, then count cells. Collect the
volume of cells required for the assay and centrifuge again. Optimal cell densities must be established for
each cell line and plate type by experimentation. Cell viability should be >90%. Resuspend the cells in PBS
or serum free media to the predetermined cell density and dispense. Up to 0.5 mM IBMX may be added to
the cell suspension.
CELL PREPARATION FOR ADHERENT CELLS - Harvest and resuspend cells in complete growth media at the desired
concentration and seed cells in the microtiter plate. Optimal cell densities must be established for each
cell line and plate type by experimentation. Incubate seeded plates overnight at 37°C. For the assay,
-6 6
RFU
CELL-BASED ASSAY PROTOCOL
6000
cAMP Standard
EC50: 26.8 nM
S:B 31.3
4000
2000
0
10 -10
10 -9
10 -8
-7 -
10 -7
cAM P [M ]
7
10 -6
10 -5