Questions related to Ecm
I have extensive 2D and 3D spheroid cell culture experience and I am considering a multi-cellular culture whereby I layer cell types on top of each other, for example stromal fibroblasts followed by epithelial cells.
Is it as simple as timing the addition of the second layer (the top) of cells addition so that both layers become confluent at a similar time, providing I use the correct mix of media and supplements for both cell types?
Or is there a little more to it such as an ECM coating of say fibronectin to stick the second layer to the first layer?
I am currently looking for an easy way to track teacher displacements in a classroom. I know you can do it with cameras and triangulation but what about new technologies? Would it be possible to do it with only a smartphone?
Thanks in advance for your time
I'm wondering about media usage in iPSC culture. Most papers/protocols work with mTESR1, but for me it seems that PluriSTEM needs less media change and no overweekend change.
Does anyone use PluriSTEM with matrigel/ECM and has good results?
and btw: Does EDTA-solution or EDTA-based solution (like Versene) for cell passaging also work on matrigel/ECM?
I follow this guide to plot CUSUM after ARDL:
But i read some journal article that ECM model is to be plotted. Can i know is it different to plot ARDL model itself or the ECM version? Which should we plot?
Cardiac tissue ECM showing more stiff nature in comparison to other tissue, it isn't solubilize easily. Is there any way to solubilize it. I have tried Pepsin/ HCl solution for digestion but it isn't working.
Hi, I want to quantify the amount of collagen(s) that got secreted outside of cells to ECM through Western Blotting the media, however I'm not sure which housekeeping gene I can use in order to normalize my data. Any ideas?
Energy Conservation Measure (ECM) analysis is a process that involves identifying and evaluating potential opportunities for reducing energy consumption in a building or facility. The objective of ECM analysis is to identify cost-effective solutions for reducing energy consumption, improving energy efficiency, and reducing greenhouse gas emissions.
Hi scientific community!
I was hoping to get some input on a problem I am currently having deciding how to fix my samples for AFM. I am using AFM to detect stiffness of the ECM in fibrotic areas, and have the option to leave my samples unfixed (we have tested that this works), or go for PFA, NBF or a solvent method like methanol.
Since we know methanol will dehydrate the ECM and PFA or NBF will cross-link it, we were intending to go straight for measuring the unfixed tissue, since this would be most "correct". However, whilst the main structural proteins like collagen will be fine for the time period of the AFM experiment if kept cool, it has come to my attention that other smaller, less stable ECM proteins could degrade if unfixed, which could affect the integrity of the fibrosis and therefore the resulting stiffness measurement.
Most previous literature either uses unfixed tissue or PFA.
What seems the best method to you - to fix the ECM, or to go ahead unfixed?
We have performed an experiment where the recombinant protein was allowed to bind to the ECM.
The ECM components was kept at a fixed concentration thereby variation with the antigen concentration was applied.
I want to find the dissociation constant for the protein.
anyone knowing please help me out..
Does anyone know about a chemical or drug, whatever that increases basement membrane stiffness ectopically? In particular, I would like to culture a tissue ex vivo and follow tissue dynamics after stiffness alteration (something contrary to Collagenase treatment).
I am looking for a summary/review of overall different integrin isoforms' affinity to different ECM ligand isoforms。 eg. integrin a7b1 has high affinity to Laminin a2, while a3b1 and a6b1 has high affinity to Laminin a5. Not just laminin but also different collagen. I have came across some papers but there should have been some previously done review.
We use ECM to predict Dutch yearly traffic kilometers. For year 2020, the covid-policies impact the traffic performance a lot. We need to use data from 1983-2020 to obtain coefficients of independent variables, such as GDP, population, oil price...to estimate traffic kilometers for year 2021 to 2025. Therefore we add a dummy variable to present the covid-policies for year 2020. The question is: should the dummy for both short and long terms or only for short term?
I am running ARDL equations for the connection between imports (dependent variable), domestic growth and the real exchange rate (explaining variables) of several countries. Now, to test for cointegration, ARDL requires the bounds test. However, I am asking myself if the following is possible: run the ARDL, then run it in its ECM form. If the error correction coefficient is significantly negative (between -1 and 0), we get confirmation that the ARDL is cointegrated and fine. Hence, it would not be a test for cointegration before running any regression but running the ECM to test for cointegration. Is this valid?
I am working on EMT-invasion of cancer cells. I have observed changes in the expression level of ECM-associated genes. Upon submission of a manuscript, one reviewer suggested: " to investigate possible changes to the organization of cell-culture ECM and its ability to support cell migration or invasion".
Can anyone help me to understand this comment? How exactly (specific experiments) can we proceed? What is it that the reviewers want?
Thanks in advance,
I am working on reprogramming fibroblasts to neural stem cells. After the NSCs induction, I am feeling it difficult to detach the cells from the surface. Even after incubating to 0.25% trypsin/Accutase for around 10 mins, the cells are not lifting off the surface. Only ~50% of the cells can be harvested and toxicity is seen because of the exposure time to the enzymes. I have also tried tapping the dish but it's not working. I can see some fibre-like structure around the cells which I think is ECM and that is inhibiting trypsin activity.
I'm currently working on decellularizing ECM produced by fibroblasts to be used as base for cancer cells culture. The problem is that even with the gentlest wash the dECM still comes off the surface easily and result in a solution containing broken dECM. I'm thinking that for the purpose of my research I can also centrifuge the solution, collect that broken dECM and embed it into hydrogel for use. Does anyone has similar experience and know if it's feasible and if so what would be a good centrifuge setting to use? Or if you have better tips on how to make dECM stay more firmly on a well plate surface you're welcomed! Thanks
tapping mode tips are too stiff to measure extracellular matrix roughness. I am looking to find the right cantilever type with a stiffness (k-value) that is able to do so in solution.
Thank you in advance
I am looking for an antibody to stain human kidney ECM Collagen III with IHC-P. I have tried using abcam's ab7778 unsuccessfully, and it is difficult to find other antibodies in the literature for this. Does anyone have any recommendations?
I want to quantify the collagen content containing intracellularly and that secreted outside the cells within the ECM. To do that, I am looking for a validated protocol to separate the two fractions without altering the integrity of the cells and the tissue. Most of protocols talk about decellularization step. Is there any other efficient technique that can be useful to separate cells from ECM fraction in order to quantify the collagen content within two fractions separately ?
we isolate decellularized ECM from various organs and produce hydrogels of these. I am looking for protocols to label ECM or ECM hydrogels with fluorophores, biotin, digoxigenin or otherwise in order to monitor e.g. degradation / turnover in time. What would be advisable?
I thought its because of the many sulfat groups which they contain ?
So they are negativly charged in some way ?
Please correct me if im wrong.
In my model, I had four I(1) variables and after running JJ test, I got the result that there are 3 co-integration equation.
Now, I want to run ECM Model, but I am not sure what to put in the Co-integration rank?
If the pore size/porosity of human skin ECM (decellularization) is around (68.3 ± 5.8)% (Wang et al., 2015), there are other results as well.
Does an artificial skin graft need a similar porosity or higher? I personally think higher porosity is the logical approach since a much more needed space for cell and ECM (collagen, elastin, etc.) growth compared to ECM (only cell grows and a bit of ECM maybe)>
I found different ways in estimating long-run and short-run when processing time series data through ARDL-ECM method in Eviews 10.
Someone use OLS method to estimasi long-run and short run, however, others directly use output ARDL for long run, and ECM for short run.
In your opinion, which one between these two method shoud I use, and why?
I want to solubilize decellularized ECM particles in order to get a homogeneous solution.
As I have seen in the literature, a common way to digest dECM is using pepsin/HCl. To do so, I used Hcl 0.01 M and 10 mg/mL dECM in 1 mg/mL pepsin at 25 C . once again the Hcl, dECM and pepsin respectively in proportion1,5,2(0.2 ml, 1mg, 0.4mg) were used (at 37 C).But I didn't get the desired result and the particles of the matter remain insoluble.
I would like to analyse decellularized ECM for an experiment, however, so far I have not been able to successfully remove the cells while keeping the ECM attached to a 96-well plate.
Protocol: Cancer cell lines were cultivated for three weeks in a polystyrene TC treated 96-well plate, coated with 0.2% gelatin. For the cell lysis, I have used two different buffers either containing 1% Triton-X-100 or 1% NP-40 detergent. I have changed several parameters for the lysis step: temperature (4°C, room temperature or 37°C), time (15 minutes up to 1h or over night) and percentage of detergent (ranging from 0.1-1%), but the ECM always detached after this step. I also added all the solutions with a lot of care.
Does anyone have any experience with decellularization in a 96-well plate and somehow solved this problem? All inputs are greatly appreciated and thank you very much in advance for your help!
I am trying to calculate an ECM with panel data and thereby I have the following problem. I run the ecm command from the ecm package an error occurs saying “non-numeric matrix extent “. I found out that the reason is the creation of the panel data. I tried two different approaches to fix the problem.
At first I created the panel dataset with the pdata.frame command from the plm package.
p.dfA <- pdata.frame(data, index = c("id", "t"))
where “index” indicates the individual and time indexes for the panel dataset. The command converts the id and t variables into factor variables which later leads to the error in the ecm command.
Secondly, I created the panel dataset with the panel_data command from the panelr package.
p.dfB <- panel_data(data, id = "id", wave = "t")
where “id” is the name of the column (unquoted) that identifies participants/entities. A new column will be created called id, overwriting any column that already has that name. “wave” is the name of the column (unquoted) that identifies waves or periods. A new column will be created called wave, overwriting any column that already has that name.
This panel_data command also converts the id variable into a factor variable. So, the same error occurs in the ecm command.
If I transfer the factor variables back into numeric variables, I lose the panel structure of the dataset.
Could someone please explain to me how to run an ECM with panel data in R?
Thank you very much in advance!
Sample R Script attached
In solubilization of decellular ECM process, after dissolving the decellular powder in pepsin and then adjusting its pH and lyophilizing the scaffold, as soon as the scaffold is placed in the crosslink solution, does the scaffold disintegrate?
I ran an Error Correction Model, obtaining the results depicted below. The model comes from the literature, where Dutch disease effects were tested in the case of Russia. My dependent variable was the real effective exchange rate, while oil prices (OIL_Prices), terms of trade (TOT), public deficit (GOV), industrial productivity (PR) were independent variables. My main concern is that only the Error Correction Term, the dummy variable, and the intercept are statistically significant. Moreover, residuals are not normally distributed, while also the residuals are heteroscedasdic. There is no serial correlation issue according to the LM test. How can I improve my findings? Thank you beforehand.
Hi, I am trying to run WB on human brain samples to detect ECM in particular collagen IV and laminins.
I have an extraction protocol and I'm doing the WB using an Iblot machine.
I am using 2 different antibodies but get not bands at all ...
the ladder is visible so I think the transfer is working.
Thanks, in advance for your help
What are the equivalent cases of cointegration trend spec. (restricted constant, constant, and restricted trend) in an panel ARDL PMG ECM setting?
I am doing a sonication study to dissolve Extracellular matrix proteins in PBS to induce gelation. I came up with a strategy to reduce heat produced by sonication and cavitation affecting proteins but I was wondering if anyone knows if cavitation itself can lead to ECM protein denaturation. Besides, ultrasonic frequencies (20kHz) can neither damage ECM proteins right?
I'm doing sonications on Ecm with PBS for 1 minute and the temperatures go up to 50 degrees. However the sample cools down in ice for one minute and goes back to 15 degrees. Is this detrimental for my ecm proteins? What temperature is bad for the ecm?
As part of my research I am trying to achieve non enzymatic gelation of ECM hydrogels by using sonication with a probe. I have been doing some samples and they all become clumpy no matter what the time and power. Any tips or things to try? I use a 10mg/ml ecm concentration and PBS
I am trying to fix MDA-MB-231 cells for confocal microscopy. The cells are seeded in PEG based scaffold using collagen based cell seeding matrix that acts as ECM. I will be imaging in hopes to find the shape of cell spheroid formed in the scaffold.
Number of cells seeded - 2 to 2.5 million
I need the fixed cells to survive transportation since I will be travelling to a different facility for confocal imaging. I need them to survive or retain morphology after fixing for atleast 24 hours.
My aim is to find out the significant relationship between FDI and its determinants. I am using bound test and error correction model.
Hi! I want to mix stationary and non stationary panel data in an error correction and for cointegration. In a non-panel data setting, this is done with ARDL ECM, where a significant EC and long-run coefficient indicate cointegration, and where a bounds test can confirm this. Now, in a panel setting, is the corresponding ARDL ECM with PMG (Pesaran et al. 1998) valid? To test for cointegarion, I can use the Westerlund test in the depvar is non-stationary but indepvars are statioary, right?
Problem in ARDL "EViews".
After running the ARDL model, we did a bounds test to test for cointegration and we found that the variables are actually cointegrated at 10% significance level. Then, we ran an ECM (Error Correction Model) test to see the short run and long run coefficients but the problem is that some variables are dropped in the short run coefficients.
Knowing that our dependent variable is logWAGES and our independent variables are logFDI, secondary enrollment rate and unemployment rate and also a dummy was added taking value 1 at 2005.
When a variable enters the main (i.e. the unrestricted) ARDL at zero (0) lag, it subsequently fails to appear in the short-run error correction model (ECM). Thus, only those variables at 1 or more lags appear in the short-run ECM. I am yet to come across any theoretical or technical reason/explanation for this.
Actually, I am looking for different terms and vocabularies of Engineering Change Management such as engineering change request (ECR), engineering change order (ECO), etc., that are available in both English and French. Thank you in advance. #ECM #PLM #BIM
Why sometimes the results of ECM are a little different from those of Breitung and Candelon’s (2006) spectral Granger causality?
I am trying to dissolve decellularized ECM tissue, and have not succeeded.
Have tried ultrasonication, HCl, acetic acid, RIPA, none succesful so far.
Any adittional ideas?
would like to dissolve for assays, so do not want to use pepsin.
Thank you in advance
I am trying to estimate the long run relationship between fiscal policy an economic growth, what is the best way to deal with reverse causality besides finding an IV, in a time series approach (ARDL, ECM, VAR).
As a researcher in the field of non-conventional machining processes, such as EDM, ECM, PeP, and the hybrid machining processes that can be derived from them, I would be interested in which of these processes you see as the best option for post-processing additive machining processes. It would be important that the advantages and disadvantages are shown.
I am interested in looking at possible alterations in ECM composition in a mutant mice while comparing the same with WT mice. Any recommendations on the best method that can give an overall compositional changes in the ECM is highly appraciated
So at the moment I am growing my VSMC on collagen coated hydrogels and measuring the the mechanosensory effects produced by the hydrogels of varying stiffness. However, I thought it might also be interesting to see whether the ECM itself produced by the VSMC would be altered depending on the stiffness of the hydrogel its grown on. However, I am unaware of any such methods that have been published about. Any ideas on how to achieve this? Any input would be greatly appreciated! Many thanks :)
I am currently trying to estimate the effect of energy crises on food prices. Given the link between energy and food prices, I am inclined to reason that ECM will be best to estimate the relationship between food price and energy price (fuel price). Additionally I would like to include dummy variables in the model to estimate the effects of periods of energy crises on food prices. This I know is simple to do.
Where am confused is, how to model price volatility in the context of an ECM. I am only interested in the direction where fuel price, as well as the structural dummies for energy crises influences not just the determination of food price, but their volatility as well.
Is the collagen a dipole merely because the peptide is a zwitterion, with NH4+ on N end and COO- on C end, or is it because of its (Gly-x-y)n amino-sequence? If it's related to it's amino-sequence and the triple helix, how is it related?
Is it just simply that the N end is the + end and the C end is the - end?
Is this dipole property only occurs when pH = isoelectric point ?
What are the Differents between "DecBM " and "ECM of a bone" and bone allograft?
Does bone allograft include "ECM" and "DecBM"? What are the differents between them?are their applications the same?
+DecBM:decellularize bone matrix
+ECM:extra cellular matrix
I am planning on performing a western blot for Lysyl Oxidase (an extracellular matrix protein that is about 30 kD). I am looking to extract proteins from lung tissue and need a suggestion for a lysis buffer that can soluble my protein of interest (ECM proteins).
I've been decellularizing 2D cell cultures for immunos and LC-MS/MS using 20 mM NH4OH. Basically, I incubate my cells with this solution for 5 min and keep the ECM that is attached to the dishes. For immunos is quite simple, as I basically fix it in PFA and it's ready to go. For LC-MS/MS I used an SDS-PAGE sample buffer and scratched the samples with it to recover as much as possible. For these assays, no problem at all.
Now I want to make hydrogels out of it and for that I need to solubilize them first. Here is where my problems start. I've tried incubating the plates with 2M urea buffer in a 0,15 M NaCl 0,05 M Tris HCl pH 7,4 buffer with 1% protease inhibitors at 4 ºC in the dark for 1h (scratching the plates before and after incubation) but haven't been able to get any protein when quantifying it (BCA, I've checked and this kit is compatible). Is there any tip/suggestion you could share with me? Is there something I'm doing wrong? Any help is deeply appreciated :)
Note: I'm now going to try and leave the samples at 4 ºC for 2 days to see if that helps.
Thanks a lot in advance!
I have an ARDL error correction model, in the CUSUM chart as far as I diagnosed there is a negative trend, moving means. Before that, I found that the explanatory variable and the dependent variable are not integrated to the same degree. I know I could not use ECM when there is no cointegration, I don't know is there any other model that gives more consistent results.
Could anyone offer a remedy?
Healthy human tissues are composed of cellular and non-cellular components, termed the extracellular Matrix (ECM), which is the perfect natural microenvironment for all our cells (for millions of years).
Human ECM is composed of over 300 different proteins with different functions to cells, which are not all identified/characterized, however finally, the ECM orchestrates our tissue fate.
Human ECM proteins are identical in humans, where as NON-human ECM proteins may provide immune reactions in humans (as often observed in clinics). – Human ECM is also a clinical safety issue!
Human placenta is (1) a waste material, (2) available in consistent quantity and quality, (3) with a very dense blood vessel system, and (4) it provides many organ functions during the entire pregnancy– all in one...Human placenta tissues were used in medicine for a very long time...
However still, there are only limited numbers of publications/teams working with placenta ECM tissue for TERM. Is this a regulatory issue? A stem cell - hype issue? Other?
Generally, there would be sufficient human placenta tissues available for cost-effective and fully human TERM approaches in a way greater scales...
I carried out ARDL bounds test which shows cointegration but my long run coefficient is insignificant. when i generate the ardl-ecm however, these are significant. what do i conclude on cointegration? should i interprete the ecm and or do i need to use another method?
Hi, I ran unit root test and found that all variables are stationery at their levels. I then tested for co integration and it was confirmed. Then I ran ARDL and ECM models. The results were satisfactory and the ECM passed all the model diagnostic and residual tests. I read somewhere that there is no need for ECM if the series is stationery at level.
I am working on a uni project and I am relatively new to econometrics.Basically I am looking for the relationship between stokc prices (lnSI), interest rate (lnIR) and ecxchange rate (lnER) over a period of time.
These are the steps I am following:
1. A simple correlation test
2. checking if they are stationary. They arent on level but the first differences are (so I(1))
3. Checking of they are cointegratted (with the Johansen test) They are cointegrated so I can procceed.
And I am a little bit stuck here. One of the papers suggests a two step ECM approach. Basically my model is: (delta) lnSI(t) = β(0) + (sum) β(1)*lnSI(t-l) + (sum) β(2) * lnIR (t-l) + β(3) * ECT(t-l)
same for lnER as well and then the other way around. I am working in stata the code I use is egranger lnSI lnIR,ecm. From the results if lnIR-LD. is significant then there is a short run effect if the _cons is significant then there is a long run effect.
Not sure if this is correct.
The other method people use in the literature is VECM I can run it on all 3 variables but I am havving difficultires to read the result. Which one is the long and short term effect of each bariable on lnSI? Also for short run I guess its the L1 values but that differes from the values on the ECM test.
Why one is correct?
Furthermore, I would like to run a impulse-response chart as well but I need a VAR for that. Can I run it on a VECM? if yes how?
Hello dear friends!
I am looking for the long relationships between exchange rate fluctuations and balance of payments. I have time series data for the variables of the model. Variables are BOP, REER, Interest rate, M3, Openness, Log Real GDP, Log gov expenditures. All are stationary at 1st order, except interest rate and BOP which are I(0). Bound test shows there is cointegration. In stata, I run ECM to see long run relationship, the problem is that the shown magnitude of the coefficients are very large. I don't know what is the problem? Can anyone help me?
Or more specifically, am I right in running such model? Or I should change the estimation method? Thanks
I want to use fibroblasts to synthesize ECM scaffold for wound care. Is it better to use embryonic fibroblasts or adult fibroblasts? And why?
I'm trying to study ElectroChemical Migration (ECM) on the surface of PBCs. Essentially a metal is oxidized at the anode, and migrates under a voltage to the cathode and forms a dendrite. The migration process occures in the aqueous monolayer under humid contitions (around 20 molecules thick). I am wondering if you can do CV under these conditions to study the kinetics of the reactions and/or measure the diffusion of the ions involved?
I'm estimating an ECM for panel data and I found X and Y are cointegrated (from various panel cointegration tests and the significant error correction coefficient in ECM). However, when estimating the long-run relationship between X and Y, the FMOLS, DOLS and the long-run coefficient in ECM were insignficant. Is there a possible reason for such results?
I'm trying to set up immunofluorescence of various ECM components, and I've tried an antibody against Heparan Sulfate Proteoglycan (HSPG) which didn't work, even after harsh antigen retrieval.
Also, general tips for ECM immunostaining in the brain would be helpful.
I am currently working with integrins. I wonder that if there any differences in terms of the staining pattern of integrins, when you use no ECM coated coverslips and fibronectin-coated coverslips.
Hope anyone with more experience than me are able to help me!
I´m using single equation modelling to find a model for the mortgage rate (endo) and the money market rate (weakly exo). I have found that both of the rates are I(0), and that the long-run relationship also is I(0) by using the Bounds-test in Stata. The bounds-test is done with out the dummies which I used to get a well specified ARDL.
I have to questions:
1. Do I have to use the same number of lags and dummies for both rates when re-parameterizing the ARDL to an ECM?
- When I use the same lags an dummies in both the ARDL and the ECM, the standard specification tests in PcGive does not give the same answer.
2. Do I need to use the same dummies in the ADF-test for the respective variables as I use in the ARDL and ECM?
/Mette Marit :)
I am trying to isolate a specific cell in the eye but I don't know how to digest the matrix components without lysing or destroying the cells. The matrix is primarily made of collagen, GAGs and proteoglycans.
I grow cells on Silicon substrates as part of my research. However, i usually throw out the substrates afterwards. I was wondering if there is a way to clean them in order to re-use them, but without changing the micro and nano-scale topographies.
I tried alcohol or trypsin (which we use to remove cells) but it did not work (i can see that there is GFP signal after cleaning, and possibly lots of ECM proteins). I was wondering if there is a protocol you use.
I would like to know if it's possible to enhance mechanically an ECM with PCL (knowing it's actually hydrophobic) without it forming an obvious biphase. Perhaps adhering electrospun nanofibers or using lyophilization.
It's a particular ECM protein that I can detect in the media well enough, especially if I concentrate the media samples. However, analyzing the concentration is more difficult due to the fact that I couldn't build the standard curve and therefore measure protein levels in the media using coomassie. Do you have recommendations for other ways to ensure equal loading of protein in samples?
The usual ECM suspects tend to be the larger companies (OpenText, Microsoft, IBM, EMC, Alfresxo, Hyland, Nuxeo etc)