Adenosine 3′,5′-monophosphate (cAMP) is a cellular added agent with axial appliance to pharmacology, corpuscle biology, and biochemistry teaching programs. cAMP is produced from adenosine triphosphate by adenylate cyclase, and its assembly is bargain or added aloft activation of abounding G protein–coupled receptors. Therefore, the altitude of cAMP serves as an indicator of receptor activity. Although there are abounding assays accessible for barometer cAMP, few are acceptable for ample chic teaching, and alike beneath assume to accept been acclimatized for this purpose. Here, we call the use of bead-based ALPHAscreen (Amplified Luminescent Adjacency Homogenous Assay) technology for teaching a chic of added than 300 acceptance the applied aspects of audition arresting transduction. This technology is applicative to the altitude of abounding altered signaling pathways. This ability is advised to accommodate a applied adviser for advisers and a advantageous archetypal for developing added classes application agnate technologies.
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The cellular added agent adenosine 3′,5′-monophosphate (cAMP) is accordant to pharmacology, corpuscle biology, and biochemistry teaching programs. Activation of abounding G protein–coupled receptors (GPCRs) changes the assembly of cAMP: Those receptors accompanying to Gαi arrest its production, and those accompanying to Gαs actuate its production. Phosphodiesterases arbitrate its degradation. Altitude of cAMP serves as a advantageous indicator of receptor activity (Fig. 1). It is frequently abstinent for screening abeyant new drugs that affect the activity of these receptors or proteins complex in its amalgam (adenylate cyclase) or abasement (phosphodiesterases) (1).
Principle of ALPHAscreen cAMP assay. This schematic of the appraisal was acclimatized from (3). This appraisal is able at barometer the activity of both Gαi- and Gαs-coupled GPCRs. Gαi and Gαs subunits act on the cAMP alleyway by, respectively, inhibiting or activating adenylate cyclase (AC), an agitator that catalyzes the about-face of adenosine triphosphate to cAMP.
Although there are abounding assays accessible for barometer cAMP (1), few are acceptable for teaching ample classes or accept been acclimatized for this purpose. Here, we outline a lab class, advised to acquaint acceptance to the altitude of cAMP, which provides a accordant applied chic that complements the address material. We acclimated this chic with a beforehand of >300 students, disconnected into classes of ~60. The chic continuance was 3 hours.
The Perkin Elmer appraisal arrangement alleged ALPHAscreen (Amplified Luminescent Adjacency Homogenous Assay) enables rapid, high-throughput apprehension of proteins, such as phosphorylated kinases (2), and added arresting transduction molecules, such as cAMP (3). We accept ahead appear the use of this appraisal for barometer phosphorylated extracellular signal-regulated kinases 1 and 2 for teaching purposes (4). The assumption of the specific appraisal acclimated in this lab is as follows: Back acceptor chaplet conjugated to an antibiotic that recognizes cAMP and streptavidin-coated donor chaplet are brought into abutting adjacency by the attendance of biotinylated cAMP, ablaze is emitted. cAMP, such as that from a corpuscle lysate, will attempt with the biotinylated cAMP and abate the emitted light, advertence that the ambition has been detected (Fig. 1).
The aim of the agreement performed by the acceptance is to actuate the aftereffect of an agonist of a Gαs-coupled GPCR on cAMP assembly in the attendance or absence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Afore the allocation of the lab that the acceptance perform, beef that endogenously accurate a neuropeptide receptor [the calcitonin gene–related peptide (CGRP) receptor in SK-N-MC beef (5)] are apparent to animal αCGRP in the absence or attendance of IBMX. Control beef are additionally apparent to alone absorber in the absence or attendance of IBMX. The beef were afresh lysed application a simple lysis buffer. Details for alertness of the corpuscle lysates appears in the “Reagents” section.
In accession to advancing the corpuscle lysates, the adviser should accommodate the abstracts for the acceptance to assemble a cAMP accepted ambit (Fig. 2). The abounding cAMP accepted ambit ambit appropriate in the kit instructions yields abstracts that are best fit to a sigmoidal curve. To accomplish the appraisal easier for the students, we acclaim accouterment alone the abstracts for the beeline allocation of this ambit to the students. Afore the lab, the samples were adulterated to ensure that the abstracts fell aural this range.
Sample of a cAMP accepted curve. cps, counts per second; [cAMP], absorption of cAMP.
Due to the bound chic time (3 hours) and the ample cardinal of students, the adviser able the corpuscle lysates. In classes with beneath acceptance and best or added chic time, the lab could be acclimatized to acquiesce the acceptance to betrayal the beef to the altered altitude and adapt the corpuscle lysates. The acceptance could additionally accomplish the appraisal application the controls to access the abstracts for the accepted curve, rather than accepting the abstracts provided to them. The attributes of the cellular dispatch is absolutely at the acumen of the instructor. For example, any GPCR that modulates adenylate cyclase activity could be used. Furthermore, the lab could be acclimatized to appraisal the acknowledgment of the beef to agonists, antagonists, or added modulators. Alternatively, altered ALPHAscreen kits could be acclimated to admeasurement added cell-signaling molecules. Accordant questions can afresh be formulated accordingly. As with our added chic (4), acceptance responded able-bodied to actuality accomplished technology that is applicative to a career in biotechnology, corpuscle biology, or biologic development.
We alpha the chic with a 15-min presentation (see added materials) analogue the use of technologies such as ALPHAscreen in biologic analysis and their advantages over added techniques. The presentation additionally includes considerations for high-throughput assays (slides 6 and 13), applied tips for assuming the procedure, and explanations apropos the best of accessories and supplies, such as the use of a white multiwell bowl for this appraisal (slide 18). During the presentation, the students, who assignment in pairs, accept aliquots of corpuscle lysates. At the end of the presentation, acceptance performed the appraisal to admeasurement the bulk of cAMP in their samples. Once the acceptance had pipetted their samples into their plates, the lighting in the chic was dimmed and the chaplet were distributed. We adjourned their compassionate from the adviser presentation with a abbreviate assorted best quiz (10 questions) during the 30-min evolution time of the assay.
ALPHAscreen cAMP appraisal kit (6760625M, Perkin Elmer)
Note: For a 360-student class, we acclimated the 10,000 appraisal point kit.
Note: These were able in beforehand by the instructor. SK-N-MC beef were seeded into T75-cm2 ability flasks at an according corpuscle body and developed in Dulbecco’s acclimatized Eagle’s average (containing 8% fetal bovine serum and 5% v/v penicillin/streptomycin). Afterwards 48 hours, back beef were ~50% confluent, the advance media were removed and replaced with beginning average [5 ml of prewarmed serum-free media, supplemented with 0.1% bovine serum albumin (BSA) with or afterwards 1 mM IBMX] for 30 min at 37°C to abbreviate the basal bulk of cAMP. Afterwards the incubation, 5 ml of average (with or afterwards IBMX), in the absence or attendance of animal αCGRP (final absorption of 10 nM), was acclaim added to the flask. Afterwards a 5-min evolution at 37°C, the average was removed and the beef were lysed with 20 ml of lysis absorber (0.3% Tween 20, 0.1% BSA, 5 mM HEPES, pH 7.4). The flasks were incubated with affable agitation at 4°C for 10 min. Samples were afresh disconnected into 100-μl aliquots in 200-μl microfuge strips for accumulator at –20° for up to 14 days.
Donor and acceptor bean mixes were able as a 1-in-100 concoction in the lysis buffer. Biotinylated cAMP was acclimated at a concoction of 0.1 units per 1 μl of donor bean solution. These quantities acquiesce added assays to be performed from one kit and accept been acclimatized from the accepted Perkin Elmer agreement (6).
Note: For a 360-student class, we able 30-ml absolute aggregate (5 ml for anniversary 60-student class) of anniversary bean mix. These mixtures are light-sensitive and charge be stored abroad from ablaze (wrapped in foil).
96-well plates (1/2AreaPlate, Perkin Elmer 6005569)
Aluminum antithesis (approximately 15-by-12–cm sheets)
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Microplate clairvoyant (Perkin Elmer EnVision able with ALPHAscreen capability)
Plate sealer (Perkin Elmer Topseal-A 6005185)
Colored microfuge tubes
Note: Any blush will suffice. This ensures that the acceptance do not abash the two bean samples. Clear, but labeled tubes could additionally be used.
Clear microfuge tubes
The bank accoutrement has been apparent for a antecedent chic (4). Anniversary brace of acceptance receives the afterward supplies:
Microfuge tube rack
1 white 96-well plate
1 bowl sealer
1 area of aluminum foil
1 96-well bowl map
4 samples of corpuscle lysates (100 μl each) in baby artificial microfuge or polymerase alternation acknowledgment tubes: sample S1, 5-min acknowledgment to absorber alone (no CGRP), no IBMX; S2, 5-min acknowledgment to CGRP, no IBMX; S3, 5-min acknowledgment to absorber alone (no CGRP), additional IBMX; S4, 5-min acknowledgment to CGRP, additional IBMX
150-μl acceptor bean mix in a bright microfuge tube
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Note: This is provided afterwards lights accept been dimmed.
150-μl donor bead/biotinylated cAMP mix in a black microfuge tube
Note: This is provided during 30-min evolution period.
Container for waste
This chic chic introduces cutting-edge technologies that are acclimated in the biologic industry, as able-bodied as by bookish analysis laboratories. The chic starts with a presentation, which introduces the biologic analysis activity and the important role that arresting transduction plays in screening for new drugs (slides 5 to 9).
Many G protein–coupled receptors (GPCRs) actuate to Gαs, arch to increases in intracellular 3′,5′-cyclic adenosine monophosphate (cAMP). There are abounding methods for barometer cAMP, best of which await on antibodies that admit this atom (slide 11).
Companies, such as Perkin Elmer, accept developed appraisal systems that accredit rapid, high-throughput apprehension of cAMP. The biologic industry uses these types of assays to accomplish screening of their compounds for biologic analysis purposes. These techniques additionally activity advantages for bookish analysis and teaching laboratories because they save time and acquiesce ample numbers of samples to be analyzed in a distinct agreement (slide 13).
The appraisal acclimated in this lab is from Perkin Elmer and relies on technology alleged ALPHAscreen (Amplified Luminescent Adjacency Homogeneous Assay) (slide 12). With this assay, pairs of acceptance admeasurement the bulk of cAMP produced in beef that were apparent to calcitonin gene–related peptide (CGRP), which is an agonist of a receptor alleged the CGRP receptor. The CGRP receptor is a ambition for alleviative migraines; therefore, cAMP assays accept been acclimated to awning for new drugs that are antagonists of this receptor. The aim of this accurate agreement is to actuate the aftereffect of 3-isobutyl-1-methylxanthine (IBMX) on the abundance of cAMP abstinent in beef advised with CGRP and to accomplish a anticipation apropos its apparatus of action. Beef cogent the CGRP receptor accept been apparent to CGRP for 5 min in the absence or attendance of IBMX.
The assumption of the ALPHAscreen appraisal is based on the antagonism amid intracellular cAMP and biotinylated cAMP (Fig. 1). In this assay, there are two types of beads: donor and acceptor beads. An antibiotic that recognizes cAMP is conjugated to the acceptor bead, which captures biotinylated cAMP. Through the absorbed biotin molecule, the biotinylated cAMP additionally interacts with streptavidin-coated donor beads. Therefore, the biotinylated cAMP brings the acceptor and donor chaplet into abutting adjacency such that, aloft action by a laser, singlet oxygen from the donor bean transfers activity to the acceptor bead. This activity after-effects in ablaze discharge from the chemicals independent aural the acceptor bead. Singlet oxygen has a abbreviate half-life and is alone able to biking a abbreviate distance, ensuring activity alteration alone to acceptor chaplet that are in abutting adjacency to the donor bead. High concentrations of cAMP in corpuscle lysates arrest the affiliation amid the donor and acceptor chaplet and abatement the ALPHAscreen arresting because the cAMP from the corpuscle lysates competes with the biotinylated cAMP. Thus, added cAMP in the sample after-effects in lower ablaze discharge from the beads.
Because the ALPHAscreen appraisal relies on the assembly of a ablaze arresting and the chaplet are light-sensitive, the lights are dimmed during some portions of the agreement to anticipate the accident of arresting afore the appraisal is completed (slide 16).
Each brace of acceptance will accept four samples of corpuscle lysates, and anniversary sample will be pipetted into the 96-well bowl in leash (3 wells for anniversary sample). To accumulate clue of which samples are in which wells, anniversary apprentice brace will actualize a bowl map with the able-bodied positions into which the samples will be pipetted. Anniversary apprentice should pipette some of the samples; for example, apprentice one could put sample S1 into wells A1, A2, and A3, afresh apprentice two could put S1 into wells A10, A11, and A12.
Once anniversary brace of acceptance has completed the accomplish categorical below, the adviser will bear the samples for 16 hours and afresh ascertain the emitted ablaze arresting with a microplate clairvoyant (Perkin Elmer EnVision able with ALPHAscreen capability). Your results, as able-bodied as a archetypal set of abstracts and accepted curve, will be provided in an Excel spreadsheet.
1. Characterization the bowl on the advanced console with your accumulation number.
2. Characterization the bowl map provided with your accumulation number, and characterization the able-bodied positions into which anniversary apprentice will pipette his or her samples.
3. Pipette 10 μl of anniversary sample into the appointed wells, afterward your bowl map, and acclaim tap the bowl on the bank to mix the contents.
Note: Change your pipette tip for anniversary sample (that is, amid triplicates). Anniversary apprentice should pipette all of the samples into their own wells, as appointed on the bowl map.
4. Pipette 5 μl of the acceptor bean mix from the bright tube into anniversary able-bodied with the samples, and acclaim tap bowl on bank to mix the contents.
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5. Cover the bowl about with antithesis for 30 min at allowance temperature.
Note: The bowl charge be covered with antithesis to anticipate the sample from abbreviating and to assure it from light. This antithesis will be acclimated afresh at the end of the experiment, so be accurate not to breach it.
6. Back the 30-min evolution is complete, pipette 5 μl of the donor bead/biotinylated cAMP mix from the black tube into anniversary well, and acclaim tap the bowl on the bank to mix the contents.
7. Seal the bowl with artificial sealing band (plate sealer) by case off the cardboard abetment and cutting the sealer assimilate the plate. While captivation the bowl firmly, anxiously abolish the edges of the sealer by disturbing forth the perforation.
8. Cover the bowl with antithesis and leave the covered bowl on the bench.
Using the advice in the Excel file, anniversary apprentice should assemble a accepted ambit application the abstracts in the book for the ALPHAscreen arresting for anniversary authentic absorption of cAMP. Application the accepted curve, actuate the absorption of cAMP in the corpuscle lysate samples. For anniversary set of leash values, account the beggarly absorption of cAMP for anniversary sample and artifice these on a bar graph.
What is the aftereffect of IBMX on cAMP absorption in the CGRP-treated cells?
What is the aftereffect of IBMX on cAMP absorption in the absence of CGRP?
What affectionate of atom could IBMX be?
What are the accessible mechanisms through which IBMX exerts its effects?
Learning Ability Type: Chic exercise, agenda presentation
Context: Undergraduate lower division, undergraduate high division
Intended Users: Teacher
Intended Educational Use: Teach, plan, assess
Discipline: Pharmacology, corpuscle biology, biochemistry
Keywords: Arresting transduction, corpuscle culture, cAMP, G protein–coupled receptor
Acknowledgments : We acknowledge Scimed Bound (New Zealand) and PerkinElmer for acceptable abutment of this teaching laboratory.
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