Ecm - Science topic

If you don't want to use robot -printed matrices or microfluidics, the old fashioned way is to seed the fibroblasts in a collagen coating , then plate the epithelial cells onto the collagen. An example:

If you are interested in diffusible factor effects, cells can be seeded on opposite sides of cell culture inserts.

If the teacher were wearing an Apple Watch, then a paired iPhone will store a variety of data, including location, steps, step length, walking speed, heart rate, etc. May not be exactly what you need, but it would provide clean data.

Good luck.

Don P. (not an Apple employee, lol)

Hi,

in our lab we use mTESR1 only for very sensitive cell lines. Otherwise we use iPS Brew, which works really well and is cheaper. I even feed only every second day with that and my cells are happy. This should be tested for each cell line though.

For passaging we use 0.5mM EDTA, like mentioned before.

Good luck with your cell culture!

Selene

Both will have same answer

Hi Denis,

To do so, we usually stain the PVDF/Nitrocellulose membrane post-transfer and before blocking with Ponceau Red solution. This is quite convenient as you can just wash it with TBS-T. After that, you can follow your normal WB procedure (blocking, washing, 1ary AB incubation, ...).

Some people are also using a highly diluted coomassie/brilliant blue staining solution the same way, but I'm not recommending it as it will be hard to completly remove it using TBS-T.

Hope this was helpful and good luck!

There are several analysis tools and software available that can be useful for performing energy conservation measure (ECM) analysis. Here are some of the most commonly used ones:

The choice of which analysis tool or software to use depends on the specific needs and requirements of the ECM analysis. It's recommended to consult with an experienced energy professional to determine the most appropriate tools and methods for your specific project.

Why not do the experiment? Measure a fresh tissue, fix it (PFA), measure again.

But to answer your question: The closer you are to the living state the better, scanning in buffer would be the best. The ECM is fairly robust in comparison to other structures, I would assume that unfixed will give you the most accurate stiffness measurements. Once you aldehyde-fix biology a lot of unpredictable things happen (tissues turn brittle, proteins are crosslinked but nothing else), and solvents are even worse because a lot of material gets extracted. Also keep in mind that NBF contains methanol. And all these parameters vary depending on which tissue you're looking at. You'll have to do the experiment! ;)

For general structural preservation PFA>NBF>solvents

There are many ways that can potentially be used - isothermal titration calorimetry and surface plasmon resonance are the two that immediately come to mind.

Without knowing more details of your experiment though (is it a protein:protein or a protein:ligand interaction?) it will be difficult for people to give informantive guidance to you.

Hi Ellen,

Thank you for this helpful answer. Not absolutley sure if this will allow me to study the cell behaviour underneath the matrix without harm them. But I will try.

Hello Zonghan Gan

The following is the review article which you may be interested in.

Best.

Hello Amelia Huang ! I'm interested in your question that unfurtunately did not receive an answer. I'm doing a similar research and I wondered if I had to add the dummy variable for a year that is an outlier for the step 2 ECM (the equation in differences) also in the cointegrating long run equation in levels. May I ask you how did you fix it in your research?

Cardiac fibrosis is associated with ECM disruption. MMPs ( metalloproteases ) play a major role in C-ECM remodeling. C-ECM remodeling is mainly controlled by matrix MMPs belonging to a large family of zinc-dependent peptides that degrade almost all C-ECM components in both physiological and pathological conditions. TGFb/Smad can trigger cardiac fibrosis and increase IL-6 levels. If you wanna study cardiac fibrosis you can induce cardiac fibrosis by Angiotensin II infusion. You can also observe a decrease in alpha SMA and collagen 2 and 3. Another really helpful tool for studying cardiac fibrosis and C-ECM remodeling are the collagen hybridizing peptides. I hope you find this helpful!!!

For more information about inducing cardiac fibrosis and studying C-ECM, you may refer to the papers attached below.

Thanks for these links!

It's only one.

Hi Shravani! Thanks for your suggestion. Yes, I am washing the cells twice with PBS. I also read that for cells releasing high ECM, it is recommended to incubate in PBS for 10-15 mins before trypsinisation. I am yet to try that.

The trick with removing cells whilst retaining an intact ECM is to do every step extremely carefully. Always pipette in and out at the same position against the side of the culture dish and one drop at a time. It takes forever but it worked for me. Also, how long are you allowing the fibroblasts to produce the ECM? I remember seeing in the literature people generally waited 10-12 days but I got much better results when I waited for about 21 days (my aim was also to culture cancer cells on the ECM).

For ExtraCellular Matrix samples I would use PeakForce QNM (Hybride Mode or similar) with contact mode cantilever (e.g. soft Ultra-Short Cantilevers from Nanoworld are good candidates)

Mr a. D.

The ECT(-1)os always the lagged value of your dependent variable.

Regards

Hi Lucia Marinas del Rey, in my experience of IHC the usual culprit is generally the antibody, antigen retrieval, incubation periods, or antigen degradation due to over processing. Since the antibody you selected has some pretty good reviews, I would recommend trying out different antigen retrieval times and methods. Heat mediated antigen retrieval generally works the best in my experience, but enzymatic could be another option. Increasing the antibody incubation period may also help if you haven't tried that yet. If you can provide a bit more info on what is unsuccessful about your staining (e.g., no stain, non-specific staining, etc.) I may be able to help more. Don't give up, IHC is a pain!

Dear Anna,

If you use tissue culture, in the first step you collect cells by solution and in the second step you collect ECM by screw.

Martin C Harmsen

During degradation, the covalent bonds of the ECM protein are broken. However, the properties of a hydrogel are very dependent on the state of the water in it. You can change the state of water by adding inorganic salt, temperature. Even adding a fluorophore will change her condition. The state of the hydrogel is controlled by the energies of thermal kT and quantum fluctuations ħω.

Hello Max Knier

Yes, you are right. Glycosaminoglycans (GAGs) are highly negatively charged because of sulfate or carboxyl groups on most of their sugars. Their highly negative charge attracts osmotically active cations like sodium ions, which causes large amounts of water to be incorporated into the matrix. This results in porous hydrated gels and is responsible for the turgor that enables the matrix to withstand compressive force.

Best.

Dear Puneet.

Why not consider using ARDL as recommended @Mwoya Byaro.

Regards

يحتاج الى مختص في مجال الجلدية

I recommand to see first the intergration order of your series. I(0), (1) I(2). Usually a mix of I(0) I(1) implies the usage of ARDL. I suggest also to see model with time varying parameters. One can see this :

The pH was 2, but I will be more careful about washing in the new series. Thank you very much for your tips.

Hi, you might be able to do it by cross-linking the ECM to the bottom of wells using cross-linking agents like glutaraldehyde.

See these links please:

Thank you I really appreciate your help.

I used skeletal muscle tissue for decellularization.

The tissue/pepsin is 10 to 1 in most articles

Is it necessary to reduce the incubation time by increasing the concentration of pepsin?

thanks@

I notice the following about your specification. (1) Your inclusion of a constant (and its subsequent significance) means you allow for (and find) a trend in the real exchange rate independent of any trends in the other variables. Is that economically reasonable? (2) I assume the CRISIS variable is a zero-one dummy for time periods with a "crisis" of some sort. Apparently it is not in the cointegration vector. Why not? If it were, then I'd expect to find CRISIS differences in the error correction equation. Instead, you have it in levels. Thus you specify that a temporary crisis has a permanent effect of the level of the real exchange rate independent of the other variables. Is that what you intend? (3) You do not include the lagged difference of the real exchange rate in the error correction equation. Why not? Normally it would be there.

Do not trust to ladder only. Apply ponceau S staining and first see your protein of interest at membrane after transfer thereby primarily validate your extraction and transfer method. Afterward freshly prepare and adjust well the concentrations conveniently of your primary and secondary antibody buffers. Validate your loading concentration and imaging method by using a housekeeping protein if possible.

Emir

Hello Gabriela Sanchez

Denaturation temperatures vary for different proteins. Generally, temperatures above 41 degree C will break the interactions in many proteins and denature them. For ECM proteins temperatures above 55 degree C will cause denaturation of proteins. The degree of damage also depends upon the duration of exposure to high temperatures.

Heat denaturation of proteins is usually irreversible at temperatures higher than 80 degree C because under these conditions proteins generally aggregate after heat denaturation.

So, I suggest you break your sonication time into smaller time intervals meaning instead of 1 min you can perform sonication for 20 secs each (3 times). While sonicating place the sample on ice and after each sonication give a break for a few secs with the sample on ice. Then again perform sonication for 20 secs and continue this cycle. This will help control the temperature and prevent protein degradation.

I hope this is helpful.

Good Luck.

Why not trying with an high power Ultrasonic Sonicator bath?

BW.

Hi Dr. Subia Bano I am using ultra-low attachment plates (Corning), I want to collect my spheres and want to fix them onto a glass slide for further ICC. Could you pl guide me for the same. Thanks in advance.

Best,

MJ

Since your objective is not to test for causal link between the variables, I think you can perform the ARDL without the causality test. The Granger causality does not necessarily address the cause-and-effect relationship between the variables however, it helps to determine if one variable can predict or forecast another variable.

Hi my dear

The following link is very important for your problem

Normally when you are using some other tests like simple OLS, DOLS, FMOLS, or CCR, they show you only long-run relation. For finding the error correction term ECT you will need to run a separate error correction model, which uses differenced data with estimated lags. Here in ECM you will look for insignificant variables and delete them one by one, not all in one go. Once you start deleting the insignificant variable you might notice that the value of ECT is becoming negative and significant which is a good sign. Somehow keep deleting the insignificant variables until they left with few significant variables. why you need to delete the variables because it will make the residuals more perfect that u can check from residual graphs.

In my view, normally short-run effects are seldomly interpreted or rather in a case when the nature of variables is such that their values change on daily basis or weekly or monthly with greater impacts on the dependent variable.

In the case of ARDL normally the ECT is calculated automatically, if you have concerns that the most important variables are ignored in the short-run estimates, then try to change the lags of the dependent and independent variables and go through a trial and error process, you might come with better results.

Kindly cross-check your results of ARDL with FMOLS or CCR as well.

Malak Sherif Not yet

I believe you are using Eviews. Please see the following Eviews forum since this seems to answer your question. It seems that Eviews display the short-run coefficient for the zero-lag variables in a specific way.

I don't know such a dictionary, but you can check the book "Conduire Le Changement - Feuille De Route En 8 Étapes" from John Kotter (available in French and English); It could give you some terms and french/english expressions about change management.

Thanks Guy.

Dear Colleague,

As far as I can see, there is no single method for all the different decECMs. Depending on the species and the tissue type used or the anatomical localisation, the respective generated decECMs have different biochemical compositions. The degree of cross-linking of the different structural proteins can also be very different.

Best regards,

JS

For the long run, I don't know a well stablished method besides the usual ones (FMOLS, DOLS, GMM, IV). However, I think your question ties in the so-called "Wagner's law" literature. If you search for empirical papers on this topic, you will probably find something of your interest.

Interesting question. Hypothetically the best post process would be no post process!

WEDM is commonly used to remove parts from build plates in LPBF but I have seen an excellent patent which details synthesis of ceramics at the interface so even metals parts can be 'broken' away from the substrate.

As for the remaining surfaces - I see all of the examples you mention as being necessary in the same way that from a surface integrity and geometrical perspective LPBF parts are essentially equivalent to good quality castings. Therefore I think there is a challenge for designers to create components which take advantage of AM but are easily post processed.

We should also be mindful that for many materials (including the ones I work with) there will be a need for heat treatments to 'CTRL+ATL+DEL' the unfavourable microstructure from process. Syncing post processing with this is also an important consideration. Great question!

Hello Vinod

Methods to analyze ECM changes such as scanning electron microscopy, immunohistochemistry and zymography could be used.

Please refer to the research article below.

Best Wishes.

It has been shown that hyperglycemia induces excessive ECM formation in muscle.

Hello. I hope you are doing well. It seems to me that in this case you can use NARDL midel to check the Asymmetric Impact of price volatility besides the ECM. moreover, to check the causal direction you can apply Toda Yamamato test.

Good luck

Please look at the following below links which may help you in your analysis:

Thanks

follow and care

ECM is mainly sugars. I'm not sure how to make hydrogels out of them, but solubilising them will require breaking the long chain sugars (hyaluronic acid, chondroitin, etc.)

Well, Your CUSUM results indicate that the linear model is inappropriate to be estimated as this indicates some nonlinear behaviour. I would suggest the the use of nonliner model. I f you want to test for cointegration, use nonlinear cointegration analysis of Seo 2006 or even some new developed nonlinear cointegration analysis.

Regards,

Dr Katleho Makatjane

Business and Risk Analyst

Basetsana Consultants

Merci pour votre contribution! Exactement, c'est aussi un sujet très culturel, mais aussi les cultures changement avec le temps! Bonne chance!

Yes definitely, however the composition an differ with different cells

But till now we believe that the operation of non conductive materials is difficult and gives inaccurate result for dimensions.

Dear Muhammad,

(1) You can use simulated body fluid (SBF). SBF can be better as it is alternative soaking in calcium, phosphate and others.

(2) An another alternative method is to immerse the samples in saline or phosphate-buffered saline (PBS) solutions. PBS or phosphate-buffered saline is a buffer solution that is particularly valuable because it mimic the ion concentration, osmolarity, and pH of human body fluids. All in all, in life science or biomedical research, PBS is largely preferred while saline is more for medical usage or in fields that cares a bit more about the chemistry.

Ashish

There is no tool wear in ECM if there is no short circuit during machining. If short circuit happens, the workpiece and electrode all may be damaged.

Conluding that all the LR coefficients to be insignificant is not a good idea. Instead, you may try including new variables or changing the length of observations. Of the results do not change, then you can present all your results and argue that there are no LR relation. I suggest following the p value for the RESET test. The higher the p value for RESET test, the lower the probability of omission of other variable(s).

Btw, have you checked your independent variables for multicollinearity?

Hello,

When the variables are integrated of order zero, Johansen cointegration test is applicable. After the long run relationship is ascertained, the next is to specify the VAR and VEC model.

Usually, researchers used ARDL and ECM for variables with different order.

Hi Thomas Woodwork stationarity can be achieved through using first differences. Also, you can use some alternative data and indicators. I'm sure there are also other methods. Keep in mind, you should use original data when first differencing don't use natural log (ln).

You can take ln afterwards. But I would like to just remind you that exchange/interest rates are already rates so do not forget considering how would you interpret ln of rate.

Look at the size of your dependent variable. I'm guessing it might be 6 or 7 digits in most countries, which would make coefficients in the thousands seem sensible.

What species?

In general fetal (not embryonic) fibroblasts are preferable to adult fibroblasts as they should be able to tolerate more population doublings in culture. Murine pups at 12.5 to 13.5 dpc are ideal for fibroblast preps.

Fabrizio Guzzetta I'll look into it.

What is the indication of the (adequate) unit root tests first?

Issues of heterogeneity and cross sectional dependence have to be considered initially and then to proceed to the adequate (new generation) cointegration tests.

Amsbio now supplies the Seikagaku Corporation 10E4, JM403, 3G10 epitopes. I use the 10E4 and it is absolutely fantastic for IF.

You will observe completely different staining patterns, depending upon the antibody you use and what method you use to persuade your cells (whatever these might be) to attach to a non-coated surface. Different cell types express and use different integrins, but all cells/integrins do require a ligand to act as the recognition and attachment target.

No coating > no ligand > no attachment > no integrin organisation!

What are your coverslips made form? Glass? Polystyrene? Thermanox? Charged/Non-charged?

What are you trying to demonstrate / observe?

Can't be more specific because you provide too little information.

In the first case the answer is a resounding no. But your analysis may reflect the fact that x or y use more lags and the converse is tractable if you are convinved that it is not mis specified. Then you can compare both with theoretical models. The one with less lags is probably more tractable because it is structurally intuitive.

In the second case the answer typically is more lags in the ADF test as the ADF tet will recommend the number of lags based on the Augmented Dickey Fullerson results. Your answer should go with what is theoretically more tractable. Also the software implementation of the test in both R and stata is typically fully automated and you need not tweak any settings apart from understanding the output and impacts on your model, if any.

Here you are

Hi Sara,

I think that the piranha solution is an effective way to clean silicon substrates and remove organic materials from the surface of the substrate. The solution is prepared by mixing sulfuric acid and hydrogen peroxide with 3:1 (v/v). Please put sulfuric acid in a beaker first. Then, put the silicon substrate in the piranha solution for 10 minutes, and rinse it for 30 minutes with distilled water.

Best,

Dear Eric Emmanuel Montes, I think you mean by ECM: Extracellular Matrix! If so, please check these data. My Regards

Thanks a lot, Engelbert Buxbaum

Hi Alberto!

Maybe this paper can help you with methodology and for reference:

http://essays.biochemistry.org/content/early/2019/08/26/EBC20190001.long