cAMP - Science topic

El avance de las tecnologías ha supuesto una gran transformación para la práctica del Trabajo Social. Gracias a estas, se ha podido sistematizar y coordinar los recursos con mayor rapidez y eficacia. Facilitando a los profesionales la organización de los servicios y la posibilidad de gestionar grandes sumas de información. Además, con la incorporación de diferentes herramientas tecnológicas es posible recopilar toda la información de los usuarios en una misma plataforma, optimizando así, los procesos administrativos. A su vez, se facilita la comunicación entre los miembros de equipos interdisciplinares para desempeñar sus funciones de manera correcta.

Proporciona también la capacidad de trabajar de manera remota, lo que resultó de gran utilidad en situaciones especiales, como en la pandemia del COVID-19. Cabe añadir que, en profesiones como la del trabajo social es necesario estar informado sobre la actualidad, por lo que el desarrollo tecnológico ha sido fundamental en este aspecto. Y por último, ha sido de gran utilidad para dar mayor visibilidad a esta profesión mediante la difusión de profesionales por diversas redes sociales.

El proyecto del CEIP Lluís Vives es un excelente ejemplo de cómo la interdisciplinariedad y el uso de medios audiovisuales pueden potenciar el aprendizaje, desarrollando habilidades comunicativas, tecnológicas y educativas de manera integrada. Francisco Zambrano-Romero

En los últimos cinco años, las investigaciones sobre la inteligencia artificial (IA) y la desinformación han explorado una variedad de enfoques y áreas emergentes. A continuación se presentan los temas clave tratados en estos estudios:

Estos enfoques representan algunos de los campos emergentes en la investigación de la IA y la desinformación, destacando tanto los avances tecnológicos como los desafíos éticos y sociales relacionados con la propagación de noticias falsas.

This is the typical Question that the longest river in the world could be the Nile river in Ethiopia.

Of course, some countries have river but it is historically recorded in many centuries before.

Good Morning

In Spain . Check Coleccion Telefonica

Transformaciones Sociales.?!..

Erinosn Huamani Ccerhuayo algunas ideas en el campo de la ingeniería eléctrica pueden ser:

También se hablan temas sobre programación en MATLAB y C++.

Saludos.

Elham Amini. E.A Differentiation of SH-SY5Y cells should be strictly monitored.

Read our recent study: https://www.nature.com/articles/s41598-024-63005-y

Feel free to contact us at any time for further clarification.

Based on my research and published work titled 'IVAL: Immersive Virtual Anatomy Laboratory for enhancing medical education based on virtual reality and serious games, design, implementation, and evaluation,' I firmly believe that the use of virtual reality has a positive effect on people, particularly in the context of education and training.

Our study involved developing and evaluating the effectiveness of IVAL, an Immersive Virtual Anatomy Laboratory that leverages VR and serious games to enhance the learning experience for medical students studying anatomy. The results were highly promising, with participants reporting high levels of satisfaction, success, and learning outcomes.

Through IVAL, students were able to immerse themselves in a 3D representation of the skeletal system, providing a deeper spatial understanding and retention of anatomical concepts. The immersive and interactive nature of VR technology created an engaging learning environment that surpassed traditional methods like studying cadavers or 2D diagrams.

Moreover, our findings suggest that VR simulations can potentially supplement or even replace the need for cadavers in anatomy education, addressing ethical concerns and resource constraints faced by many institutions.

I encourage you to read our paper (link: https://www.researchgate.net/publication/376621926_IVAL_Immersive_Virtual_Anatomy_Laboratory_for_enhancing_medical_education_based_on_virtual_reality_and_serious_games_design_implementation_and_evaluation) to gain a comprehensive understanding of the design, implementation, and evaluation of IVAL, as well as the positive implications of incorporating VR technologies in medical education.

Hi, usually I add the ligand then add forskolin. For Gi-coupled receptor, you may need to optimize the conc. of forskolin firstly.

Caro Anita Carpinteri

L'employer branding svolge un ruolo cruciale nella creazione di una Employee Value Proposition (EVP) di successo. Essenzialmente, l'employer branding rappresenta la reputazione di un datore di lavoro sul mercato del lavoro. Una EVP efficace deve essere supportata da un employer branding forte e positivo, poiché quest'ultimo contribuisce a definire l'immagine dell'azienda come un datore di lavoro desiderabile. Un employer branding positivo attira i talenti, riflettendo la cultura aziendale, i valori e le opportunità di crescita professionale. Quando l'employer branding è allineato con l'EVP, il messaggio inviato agli attuali e futuri dipendenti è coerente e attraente. Le organizzazioni che investono nella costruzione di un employer branding autentico e positivo creano una base solida per un EVP di successo, aumentando la loro capacità di attrarre, trattenere e impegnare talenti chiave.

Spero che aiuti.

Jennifer, a valid answer to your question immediately won the Nobel price :-)

Your question is absolutely relevant to understand how cells are functioning. Just considering GPCR, as you wrote, there are about 800 independent sequences, though more than half of them are involved in smell sensation. There is an estimation of how many different signals can be sensed by GPCR: ~30-100000. As you raised, how come that these many different inputs can be translated to proper cellular responses, regulating the expression of 20000 genes, by utilizing actually 3 second messengers: intracellular free Ca2+, cAMP, and the activation of G12/13. It is literally impossible to explain this only with compartmentalization, which might affect though the functional outcome of the original signals. But the extent of information shlould be in the same range during the whole signaling processes, so the tenthousends of inputs, which results in tenthousends of outputs, should involve tenthousends of poossibilities during the whole process. The only way it is possible is the coding of the input signals, into the numerous spatio-temporal activation shapes of the second messengers. e.g. different shapes of transients and waves of the second messengers' intracellular concentrations, which somehow integrates on the next coding on the transcription factors, etc... So, in my opinion, there is noe definitive answer to your question right now, but this is oone of the most interesting, exciting, and important problems in current biology research.

Best, Karoly

Dear Salman, the protocol describes this in detail, but for some more information see the attached files.

If you have any specific question I'll try to help.

Best: Károly

I think that this is possible through the usage of qualitative methodologies like the Loop analysis. In fact, by considering the effects of climate change on both the social and ecological spheres, it can be understood the influence of this negative phenomenon with respect to cultural heritage.

Possibly, proxies may be used, such as the native population or the number of people visiting museums and enrolling in humanities studies. However, it would be important to take into account also government incentives and plans, to avoid errors and bias.

If this is just a solubility question, you can dissolve IBMX in DMSO at room temperature. You could make a concentrated stock like this and store at -20 for later use. I usually prepare a small volume of 100mM IBMX in DMSO, then take whatever is needed to achieve 0.5mM in my buffer.

As you said, one phase exponential decay is a good curve for your data, but you should directly use log [cAMP] for x-axis. In Prism, the standard curve can be fitted in "Analyze", "XY analyses", "Nonlinear regression (curve fitting)", and "One phase exponential decay". This allows for an estimation of the parameters S (max), P (min) and k (rate) of the equation

Y=S*e^-kX+P.

This formula easily transforms measurements (Y) into concentrations (X). If you have difficulties, you can send me your data.

In the assay the cAMP you want to measure competes with the biotynylated-cAMP that is a part of reagent fluorescent conjugate. Geenerally more of Your cAMP then less signal you obtain.

https://www.perkinelmer.com/pl/product/alphascreen-camp-detection-kit-1000pts-6760635d

Read the kit directions carefully. I'm guessing the lysis buffer is merely the preferred buffer for the assay and they expect you to mechanically lyse the cells (e.g., sonication, french press, barocycler, microfluidizer, or freeze-thaw cycles). cAMP Ab assay requires that you wash any complex media from the cells before you lyse them to avoid any media suppression of the assay results. It also brings everything to the right pH to optimize the Ab assay. You can't use surfactants to lyse the cells because these will interfere with the Ab assay. My own preference is Barocycler or Microfluidizer, because they are very consistent and don't alter the temperature of the sample much (e.g., about 4 C), but they require a significant capital outlay to acquire one.

The 6-Bnz-cAMP is a membrane permeable and selective cAMP-dependent protein kinase (PKA) activator. It is a sodium salt and keeping at room, temperature overnight may not be good enough to degrade it. After noticing the issue you might have again kept it at -20 degree C. It would have been the matter of concern once it is reconstituted, hope the vial containing powder form was kept overnight at RT but not the diluted reconstituted or the diluted vial.

The company provides SDS wherein recommended information is given for the product long storage, stability with minimum or no degradation rate. Of course, you must be careful with your chemical storage as per the recommendation of the company SDS manual, to me however, it is not a great mistake that could affect the stability of the sodium salt to the extent which makes you reluctant not to use it as PKA Activator for your experiment.

Best

Hi,

According to studies on Sw620 and Sw480 cell lines, one of the best methods for transfection is to use polymer-based transfection kits. So you can use Polyethylenimine (PEI) or commercial kits such as Arrest-In reagent or JetPei.

The original poster probably no longer needs this answer, but I figured I would answer anyway, in case someone else has the same question.

When looking for agonist activity at A3 AR, it makes sense to treat with forskolin, just as the original poster said. That way, you can compare the treatments to the forskolin control and see if any of the treatments have decreased cAMP relative to control (which means that A3 AR was activated).

I suppose that one could also test for antagonistic activity at A3 AR by having a forskolin control along with a forskolin+NECA control. A successful antagonist would be able to out-compete some of the NECA and would yield a higher cAMP value compared to the forskolin+NECA control. However, in our lab we have never actually done this. We have only ever tested for agonism at A3 AR.

We designed and carried out a correlation trial among INR, chromogenic Factor X and Factor II:C to achieve some conclusions in a population of anticoagulated patients with APS. We only included a normal control population. Further, a control population without APS but taking anticoagulants should be added (a small number of participants has already enrolled).

PKA Activators | SCBT - Santa Cruz Biotechnology

pka activator | Sigma-Aldrich (sigmaaldrich.com)

Human Prekallikrein Activator (PKA) Reference standard PKA-9H - Creative BioMart

In the Spanish case, the influence of Campos Venuti (and his Urbanistica e austerità) was huge in the Spanish municipal plans of the 1980s, undertaken by the first democratic city councils for cities that had to correct enormous deficits of public facilities, infrastructure, urbanisation or housing, inherited from the dictatorship. In the 1985 Madrid Plan he was directly involved. In other cases, like Valencia, Málaga or Santiago de Compostela, his ideas had a strong influence.

A book on this Plan of Madrid was recently published. Three articles, contained in this book, written by Joan Roger Goncé, Beatriz Fernández Águeda and Sonia De Gregorio Hurtado clarify several aspects about the relationship between Campos Venuti and Spain.

Otherwise, Federico Oliva colaborated for many years in the journal Ciudad y Territorio of the Spanish Ministry of Public Works and Urbanism.

The relation between Italian and Spanish urbanism was very intense and fruitful since those years. Proof of this are the different tributes celebrated in the Club de Debates Urbanos de Madrid to figures like Francesco Indovina or Bernardo Secchi.

Dear Joss!

Please You look at these publications (attached):

Dear Sam,

The answer to your question worths a Nobel prize :-) In my opinion, this is one of the very important open questions in biology. You very clearly described the core of the problem: there can be dozens of thousand cellular outcomes as responses to several dozens of thousand extracellular/environmental stimuli - transmitted by a few second messengers. Certainly, there might be several different mechanisms in maintaining specificity. Compartmentalization and other ways of localized production of second messengers very likely contribute, but I think these classic biological processes are, by far, not enough. The missing information might be encoded into the spatio-temporal dynamics of second messenger levels, or other, as yet not identified biophysical/biochemical features of the complex interaction network in cellular signaling. So, there must be a lot to discover :-)

Cheers, Karoly

Dear ,

Thank you so much for your response. I also found some commercially available ELISA kit from Abcam. With some minor adjustments from manufacturer's protocol, the results are reliably obtained.

Drs Mullen and Tripthi,

Thank you very much. I appreciate !

Hi there,

I don't think that you can detect cAMP in a Western Blot, since cAMP is not a protein but a relatively small metabolite.

I am also not sure if it will fix well during perfusion of the animals and if it is stable during the tissue processing. Soeven if you stain against cAMP, I am not sure how well this represents the in-vivo situation...

There are antibodies against cAMP that should work well in ELISAs and probably you will find an HPLC method for the determination of cAMP in tissue lysates.

Hi Sunilkumar Shukla ,

Interesting! I have been involved in a pilot study with drug-induced memory dysfunction and actually my thesis extended further to employ scopolamine-induced AD like neuropathology and memory loss.

However, my study was mostly based on the changes in the EPSP and neuronal activity of hippocampus and cortical regions as well as behavioral assessment in rat models.

Anyways, the main issue regarding the levels of BDNF in AD subjects is that the mRNA and expression is apparently declined in hippocampal formation of subjects with AD:

Different SNPs of the BDNF also have been studied showing a direct link to AD symptoms:

VGF and its C terminal is important in formation of new episodic memories via modulation of function in the DG. This can help when you design your behavioral experiment (e.g. spatial assessment in rodents, reference or working memory testing...):

please write in English...

It was a heating problem. A new tissue processor has fixed the problem and the HistoGel is stable. Thank you

In purely mathematical sense, what you want to know/evaluate is the distance between two sets, namely a set of observed vs. predicted values. Unfortunately the distance may be defined in many ways, producing different numerical values for the same case. One of the most popular is rms deviation (\sqrt{\sum (p_i - o_i)²} / n, where n is the number of pairs prediction--observation. It is popular because there are many books dealing with this problem as well as computer programs to adjust unknown parameters in order to get possibly small rms deviation. But for calibration purposes one prefers another distance: max_i |p_i - o_i|.

Richard Premont

Dear Richard Premont

Many thanks for your response. I read the paper you suggested. What I realized was that D2 receptors involve in rewards signaling and various mechanisms which you also mentioned had been identified. D1 and D2 receptors act appose each other. For example, D1 receptors stimulate cAMP-dependent kinase, whereas D2 receptors inhibit it. The question is that which one is responsible for reward?

Sorry, no I don't know anything about this. I suppose it is possible that cAMP could get into cells randomly via endocytic vesicles, but that wouldn't be very efficient.

Following...

Stephen Boulton thank you so much for your answer! I will look at the Biophysical Techniques paper for techniques that I can try. I would like to observe cAMP dynamics in real-time, so I'm not sure if cell disruption would make this impossible (...?)

Also, I was told about a kit that could be used (R&D systems, cAMP Parameter Assay Kit), however some of our collaborators have not had much success with this one.

Thanks again!

Prof Fernando, de fato este questionamento é muito relevante e, ao meu ver, tem sido já bem estabelecido. Acredito que alguns aspectos da "dor em pontada" que carecem maior estudo, especialmente molecularmente, é o processo de transdução da informação e o envolvimento de mediadores moleculares neste evento fisiológico. Parabéns pelo tema para discussão.

Hi Pablo,

Yu's method is a possible solution to read the concentration from a standard curve linearized this way. Nevertheless, I suggest you to use your Prism software to fit the 4-parameter logistic directly, instead of the linear approximation on a log-log scale. To produce a calculation-stable fit, Prism needs the log of concentration as independent variable (X) instead of the original concentrations. Do not change the measured OD values (Y variable). Prism let you read the unknown concentration, it is a built in functionality. The advantage of using Prism (or any other similar sotfware with non-linear regression) is that you see your logistic curve, instead of just the transformed - linearized log-log values. Only the close-to-linear (middle) part of the standard curve might be used for determination of the unknown concentrations. It is rather straightforward this way, you can avoid the use of the lower and upper curving parts of the concentration-response curve easily.

Cheers, Karoly

Thank you very much for your reply.

Hi Maria Teresa,

We have been investigating executive functions for a while in young, adolescent and adult high level football players. We are using a combination of tasks that assess response inhibition, multiple object tracking, planning, working memory etc...

Happy to chat more if you would like.

Job

Many thanks Ashley,

I wil look into those. My supervisor has said I will need to subclone (and cry for two weeks hahaha) if they don't pick up.

The mechanism for hypocholesterolemia in hyperthyroidism is increase fatty acid clearance, but lipolysis is increased even more, resulting in high serum fatty acid concentrations.

Thank you very much!

Dear Anna,

so there is something that raises pCREB levels in your controls. Can it be that there is naturally a high cAMP/pCREB level in these cell lines? If so, this should be suppressable by treatment with PKA antagonists like Rp-8-Br-cAMPS (see Gjertsen et al., JBC, 1995 and some my old papers). If Rp-8-Br-cAMPS (1mM !) Pretreatment does not lower pCREB levels, CREB is phosphorylated by another protein kinase than PKA. There are some, e.g. Calcium-Calmodulin-dependent kinase, pP90rsk, p38.

All the above, however, does not solve the question why the bacterial adenylate cyclases have no effect. I suggest to contact Prof. Dr. Roland Seifert at the Hannover Medical School for this question. Dr. Seifert works with adenylate cyclases and their pharmacology. He may have an idea.

Sorry, that I have no more immediate ideas. Best wishes to you and all the best for your next experiments!

Erik

Rap1 activation assays are great but many signalling pathways including PKA can activate Rap1. I would suggest activation of Epac with 8-pCPT-AM. Nevertheless if you require semi quantative data in the way of WB, I would use PKI to inhibit PKA to fully only see Epac-dependent activation

As Pablo mentioned most people use the active-Rap1 assay for Epac1 activation. With additional antibodies this can used for other small GTPases that bind to GST-RalGDS-RBD. You can either make this protein as I did in my recent publication or purchase the kit from various places such as CST or Thermo. Rap1 is also activated by PKA and therefore I would use the super Epac agonist 8-pCPT-AM to asses if Epac is involved in you signalling pathway. Feel free to message me if you want any more information as this is very close to what I just completed my PhD on.

I used an ELISA based kit from Amersham. It was useful although a little expensive. It is not difficult but it would require a whole day and it has a very detailed protocol. I recommend it. Good luck!!!

Thank you very much. These are really useful articles.

Thank you very much for your answer! 

It seems that cAMP can move between the periplasm and the cytoplasm. See this paper:

I see a technical problem there. Probably more related to measurement than to incubations but you may check both.

You may consider to move to other more "reliable" methods (at least for us), namely HTRF  based. I copy our methodology as written in one of our papers:

Forskolin dose-response curves in different density of cells were performed to select the most appropriate conditions of the assay, which resulted in 5,000 HEK-293T cells, 7,500 neurons and 0.5 µM forskolin. Subsequently, assays were performed in medium containing 50 μM zardeverine, placing cells in 384-well microplates. This was done by the preincubation with reagents ... , followed by ....  addition (100 nM final concentration) and, after 15 min incubation period 50 µM forskolin was added. Readings were performed 15 min later using a homogeneous time-resolved fluorescence energy transfer (HTRF) method requiring the Lance Ultra cAMP kit (PerkinElmer) and fluorescence readings (at 665 nm) in a PHERAstar Flagship microplate reader

Dear Cornelius,

Many thanks for your reply. Hartmann Analytic had it.

Dear Xiao,

I think reading the below manuscript about Regulation of Lipolysis in Adipocytes could help to take off your confusion.

Regards

Ehab El-Haroun, PhD

doi: 10.1146/annurev.nutr.27.061406.093734

Dear Gerrit, 

I used a kit based on Elisa method to measure intracellular cAMP and I used samples obtained by lysis with RIPA buffer!

You can use your samples of course!

Marianna Ranieri

Hi,

GFP can be spectrally separated from CFP quite easily but will definitely bleedthrough into YFP channel. Therefore, your YFP/CFP ratio will be increased due to GFP emission and it might reduce your sensor dynamic range and sensitivity. In addition, comparison of cells with or without ShRNA will be problematic because of this. I would suggest to replace EGFP with mCherry or another fluorophore that could be easily separated from CFP/YFP.

Hope this helps,

Tal

Hi Halah,

I think the first screen, is the the use of pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). If this give the effect, you are done. However, if you need to find which PDE-isotype active, then you have to use more specific inhibitors toward various PDE type.

Good Luck,

// Beston

THANKS!!!

Actually This question you should ask to Sigma Aldrich. Anyway my experience says that these are stable in deionized water for some time. Of course we always use it as fresh solutions.

Thank you Karl and Cornelius! 

Thanks Rink for sharing your thoughts. I was also thinking in same direction and to understand better, I planned to run binding assay on same cells (whole cell binding assay) not membrane binding assay.

Do you have any suggestion in this line

I am not sure of throwing any light but what is your aim?  SEAP assay or screening delta opioids?  

If the latter, measure cAMP by RIA, ELISA or the Cisbio kit in the presence of blockers of PDE. This will enhance your throughput and let you forget SEAP.  

You can contact andreas.rennhack@caesar.de. He may help you with the compounds.

If a kinetic assay is of interest, you could try Montana Molecular's cAMP assay.  A BacMam transduction in suspension delivers a robust genetically-encoded biosensor that changes in fluorescence intensity when cAMP levels increase via Gs or decrease via Gi.  This sensor can be combined with a different red sensor (such as R-GECO) to measure two signals simultaneously.  See attached article.

Hi,

Anne and Tom are right! First you need to serum starved cells (an overnight will be enough). Second, the use of IBMX is needed to assure that cAMP are not being degradated. cAMP increase is very transitory since it is rapidly degradated by phosphodiesterases. Being so, cells should be stimulated with alpha-msh in the presence of IBMX. Moreover, duration of stimuli is also important to achieve the maximum increase of intracellular cAMP...that I think it should be at 5min maximum. I used once one kit from Applied Biosystems to measure cAMP production by MC5R stimulated with alpha-MSH (Rodrigues et al, MCE 2012)...it was a chemiluminescence kit and it worked perfectly. Good luck. 

I have never used this kit but our lab uses the this one form Cayman Chemicals and it works well.  Good luck

I think doing analysis with C18- RP columns is not the best option. Your analytes are polar (log D at pH 5.5 is -5.75 for AMP, -8.47 for ADP and -10,79 for ATP according to Chemspider), retention based on hydrophobic interaction will be minimal. Several metabolomics-articles discuss the analysis of anionic species, including nucleotides (for example Bajad et al. doi: 10.1016/j.chroma.2006.05.019). They might provide a good basis for further optimization.

Thank You Nyla for the feedback.

Thank you for your answers. We use RIPA buffer now and it seems to work.

The affinity of antibodies is always high (Kd = 10-10 ... -15 l/mol). According to the law of mass action, it's a fast reaction taking place within minutes. Limiting in ELISA is the diffusion to the solid phase ... and much more the time you need for pipetting over the plate. Therefore normally incubation times of 90 min are in use to avoid time gradients which increase the coefficient of variation. Due to the fact, that the affinties of your antibodies reacting with the antigen limit the detection limit only, the increase of incubation time wouldn't change these detection limit.

Thank you Ezequiel.. :) 

Dear Mervi, have you ever measured cAMP on stored cell lysates samples?

Jess, acording to the Annex of the Euromedex manual (http://static.bioport.cn/data/upload/product/specification/396/1342594983673_396560.pdf) IPTG (final concentration 0.5 mM) is usually added to the medium in order to induce the full expression of the hybrid proteins. So, probably without the inducer only leaky expression will be achieved and you should add the IPTG to the plates.

I hope this might help you!

Since your tissue is so small and precious, you may try to measure cAMP indirectly via its effects on PKA activation. One such way would be to do your treatment, fix the tissue with PFA, slice it and do IHC for phospho-PKA substrate (antibody from Cell Signaling). This is not specific for PKA, but gives you a hint.

There are also examples in the literature for IHC of cAMP and cGMP using antibodies against these  (http://www.jbc.org/content/272/50/31489.full) and the antibodies are commercially available (http://www.antibodyresource.com/search/Antibodies/2319c8e0-28e2-529b-9030-bdb39bb25769/camp/application/IHC-P).

Good luck!

Erik

You can get the similar or apropriate answer by searching the keyword in the GOOGLE SCHOLAR page. Usually you will get the first paper similar to your keyword.

From my experience, InsyaAllah this way will help you a lot. If you still have a problem, do not hasitate to let me know.

Kind regards, Dr ZOL BAHRI - Universiti Malaysia Perlis, MALAYSIA

According to me, using some ubiquitin affinity matrices like agarose-TUBE under stringent conditions and going for MassSpec would be the best option. Before going for Massspec, u can standardize the conditions by analyzing the effect of stimulation kinetics +/- proteasome inhibitors by western with ubiquitin antibodies.

If you prefer to do IP, antiUbiquitin antibodies will enrich mostly free ubiquitin and you may have to use polyubiquitin linkage specific antibodies. Good Luck

No, not yet, but I will! Thank you

When measuring cAMP in cells, it is very common to perform this assay in the presence of IBMX, a phosphodiesterase inhibitor that prevents breakdown of cAMP.

As adenylate cyclase inhibitors for mast cells, the antagonist against EP4 receptor and the agonist for EP3 receptor are effective.

PGE₂ inhibits the secretion of histamine and heparin via attenuated cAMP-PKA axis by binding to EP3 receptor. In contrast, PGE₂ promotes cAMP synthesis by binding to EP4 receptor.

I agree with Filipe.  Also, try spiking your lysates with exogenous cAMP as a technical control. Protease activity could digest the ELISA Ab.

Hi Maurizio, the cell number depends on the size of your culture dish/experimental set-up. There are several articles describing decidualization in vitro (for example Schutte et al. Fertility Sterility 2012: 97;997), with more experimental detail. With respect to the FBS; if you supplement with hormones you should always use stripped serum or Hyclone, otherwise you don't know the amount of hormones you are adding. Personally, I think 10% (stripped) FBS might be a bit high, because it strongly reduces the free hormone concentration in your culture.

I hope this helps, regards, Majorie