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Cell Migration - Science topic

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The Boyden chamber protocol requires a high budget for us. We want to try modifying this test instead. Can we combine it with a protocol like the filter diffusion protocol?
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If you are looking for a substitution for this experiment maybe you can try Zigmond chamber. It's resumable and can be used to monitor cell migration in a real-time manner
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hello. I am a student studying MTT assay and Wound healing.
Studying MTT and Wound healing, I learned that MTT measures cell viability and Wound healing measures cell proliferation rate and inhibition of migration.
However, I thought that it could be said that cell migration was inhibited by the inhibition of cell viability.
Can you tell me the exact difference between MTT and Wound healing?
Or do you have a paper to refer to when studying the difference between MTT and Wound healing?
thank you.
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Hello Eunji Han
The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric assay is based on the reduction of MTT to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduces the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution and the resulting-colored solution is quantified spectrophotometrically. The darker the solution, the greater the number of viable, metabolically active cells.
On the other hand, Wound Healing assay is a standard in vitro technique for probing collective cell migration. In this assay, a cell-free area is created in a confluent monolayer by physical exclusion or by removing the cells from the area through mechanical, thermal, or chemical damage. The exposure to the cell-free area induces the cells to migrate into the gap. In this assay it is essential to inhibit proliferation of cells because closure of the wound should happen only due to cell migration and not by cell proliferation. For this reason, the cells need to be starved for at least 16 hours before the scratch (wound) is created. You may provide the cells with 0.5% serum containing media which may help prevent cell death but at the same time inhibit cell proliferation.
So, MTT assay measures cell viability and is mostly used to check the cytotoxic effects of compounds under study. Wound healing assay measures the basic cell migration parameters such as speed, persistence, and polarity. Cells at the edge of the created wound polarize and migrate into the wound space. This assay does not require the use of specific chemo attractants or gradient chambers, and it generates a strong directional migratory response. It is most reliably analyzed when performed using time-lapse imaging, which can also yield valuable cell morphology/protein localization information.
In short, MTT assay checks cell viability and Wound Healing assay checks cell migration in terms of speed, persistence and polarity.
Best.
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Currently we have a two-chamber organ-on-a-chip setup of three cell types (immune cells+cell type A in one chamber, cell type B in another chamber) where we want to measure immune cell migration from one chamber to another. Currently we are measuring immune cell migration in terms of absolute number of cells migrating but in such cases, we have to image the entire setup under a microscope. Is there a scoring system for immune cell migration?
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It's relevant to have a better understanding about the context and your hypothesis to be tested here. Because you can measure migration by different ways. For example, with Boyden chamber, transwell chamber or adoptive cell transfer in a animal model. If you can provide here more info regarding the question to be answered I can be more precise. If you prefer contacting by private message you are welcomed.
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What is the best way to investigate the role of Rel B in cell proliferation and cell migration? I want to over express Rel B in these cells and study its role in hypoxia
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thanks Ilya B. Tsyrlov for the information
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I am currently doing ex vivo work using ectocervix tissue. My tissue has a lot of cells migrating out but none of the cells are attaching on the plate. The cells seem to just be sliding on the surface. Has anyone done the explant method successfully using human ectocervix tissue? Should I coat the plates or use different media? Currently using DMEM (10% FBS) and also KSFM serum free media. Both media shows no attachment of cells.
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Thanks for the answers. This was helpful.
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I am starting to investigate cell migration (A549 cell line; wound healing assay) for longer than 24 hrs. I want to make sure I am looking only on migrating but not proliferating cells.
Are there any suggestions which proliferation inhibitor I can use, which otherwise don`t inhibit cell migration?
Thanks
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A low concentration of mitomycin C may suit your experiment - here is a paper detailing the experiment in A549 cells:
Alternatively, you could use thymidine to block DNA synthesis at the G1/S phase boundary
Good luck with your experiments.
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I used Cytochalasin D as an actin inhibitor in epithelial cells. Crystals formed and blocked the whole well (96-well plate) at 1 µM concentration, and formed few crystals at 0.5 µM, and no crystals at 0.25 µM and 0.1µM. It continued to inhibit cell migration even at lower concentrations. I'm just curious, why does it crystalize.
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Dear Farah,
Cytochalasine D has a very limited solubility in water, but it is soluble in DMSO or ethanol. This is why you can prepare a solution of the drug in DMSO and add a small amount to the water-based culture medium.
If you give too much of the solution into your media, the Cytochalasine cannot dissolve, but it precipitates. Apparently, you cannot have 1 µM final concentration in media, but 0.25 µM is still fine.
Moreover, you wrote that you see an effect of the drug at that concentration, so it should be fine to continue with a maximum concentration of 0.25 µM.
Good luck,
Sebastian
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I am working on development of a tool for the analysis of cell migration and tracking. The tool could further be used for different other object tracking purposes. For this work, I am looking for a few publicly available datasets containing microscopy images of cells or small particles.
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Dear Hritam Basak,
I invit you to visit these links, to help you understand more, cell migration analysis methodology:
Best regards,
Pr. Hambaba
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Does some gene mutation that increase migration necessarily increase invasion?Is it possible that some mutation that enhance cell migration but inhibit its invasion?
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Migration is a prerequisite for invasion. Non malignant cells can migrate without being invasive e.g. neutrophiles. Invasion requires the ability of digesting sorrounding tissues. This requires the activation of metalloproteases and other proteolytic enzymes. In turn this requires an acidic extracellular matrix. As you can see, everything is interrelated.
Migration can occur without invasion. The contrary is not possible.
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I am looking into the Rac1 and RhoA antibodies as they are typical markers for cell migration. When staining the cell, which Rac1 and the RhoA is stained? Does it matter if it is Rac1-GTP or Rac1, RhoA or RhoA-GTP?
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GTP-state is an active form of these proteins. You have to see in literature whether active proteins have different cell localization. Also, it depends on your research question, what you gonna see: to compare active form in some condition and so on
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I shall be assessing the migratory capacity of activated microglia in response to treatment with a number of agents. I was wondering if there are any kits that are more preferable over the wide range of systems available.
Corning has many options: Falcon, Transwell and Corning FluoroBlok. Choosing the most appropriate is rather confusing. However, I gather that the PET membrane with a pore size of 8 um is the most commonly used parameters. However, the right type of insert eludes me.
What are your suggestions/recommendations?
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Many thanks Karina for your helpful reply. I also found a similar table by Corning, which I shall be sharing shortly.
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Hello all,
I have some immunofluorescent images of cells migrating away from a central spheroidal cell cluster. I would like to quantify the extent of this migration by measuring a specified distance from the central spheroid and calculating the number of migrating cells within that range.
For example, let's say I want to measure the total number of migrated cells within a 100 micron distance from the outer boundary of the spheroidal shape shown in the attached image. I've outlined the outer boundary of the spheroid as "1". Is there a way to then tell ImageJ/Fiji to create a shape whose boundaries extend 100 microns in all directions away from boundary "1" (ie, boundary "2")?
Unfortunately not all of the spheroidal cell clusters I am analyzing are perfectly round/circular in shape, so I don't think I can accurately measure this using a circle and some simple math. Any advice (or alternative solutions!) to this would be greatly appreciated. Thanks!
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Hi Jacob,
Check this out and it works with ImageJ:
Good luck!
@Sathya Srinivasan
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I am attempting to evaluate the interphase between matrigels after manipulating some factors in each gel.  I am having trouble visualizing the interphase under the microscope.  Has anyone had experience using dye with matrigel?  I have never used phenol-red free matrigel - does this difference in color appear under the microscope as well or just grossly?  It is quite expensive and I am unsure if it is what I need.  I am looking for a dye (or some method) to see the interphase between the gels, something that does not wash out in the cell media used to seed the cells, and something obviously not toxic to the cells themselves.  Any advice is appreciated. 
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Hi Patricia
Do you already work with the FITC-labeled protocol with Geltrex? Would be perfect for our analyses...
Thanks
Julia
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I am using the scratch/wound healing assay as a platform to study cancer metastasis. This assay should mimic cancer cell migration for metastatic tumor initiation. However, after reading many manuscripts and protocols utilizing this assay, I found conflicting information on what percentage of serum to add to the cell culture media to allow for cell migration instead of proliferation. Many papers/protocols say to use normal growth media (10% serum for most cancer cells), but others say that if serum is present then what you will evaluate will be cell proliferation into the wound instead of migration. Some papers says use serum-free media and others have used as low as 0.5-1% serum. What is the correct amount of serum to use in order to mimic the human physiological environment to study cancer cell migration in this assay?
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I have done this assay with the normal serum amount (10%) and I could not check any difference in the cell migration, comparing the treated to the control cells. I guess the cells should be FBS starved overnight before starting your assay, and it should not exceed more than 24h.
As a suggestion, there are some assays with confirm the cell migration more easily and reliably. Please, check search for migration assays using inserts. In this case, you can measure the cell migration through a membrane. Depending on the insert type, you can look at both physical migration and invasion, based on the collagen degradation.
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I performed this assay many times but without any consistent result. I usually isolate mouse spleen CD4+CD25+ cells, seed 100K non-activated cells on 3um-pore size membranes (24well plate, corning) in RPMI + 0.5% HI-FBS, with my chemoattractant in the lower chamber, 6h or overnight migration at 37 degree. The number of migrated cells is extremely varied (few hundreds-few thousands) and the results as well.
I want to try coating the membranes with fibronectin (or collagen) to improve migration. Does anyone know the optimal concentration of fibronectin (or collagen) for Tregs? I've found only few works using 10ug/mL.
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I did cell migration by cell wound healing assay in 12-well plate for HCT-116 cell line. After making a scratch, I took 2 photos per well, I got a photo with wrong phase- contrast as the attached file. Can anybody recommend for me in my protocol? Whether something wrong in the position of the scratch and phase contrast
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If you must use phase contrast, try larger wells (6 well plates). with the scratch in the center of a 35 mm well, you can avoid strange optical effects from the edge of the well. Still adjust the condenser and phase rings for imaging each well to optimize captured image quality. Also make sure to have enough medium, and the same amoint of medium in each well to avoid meniscus effects.
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I am currently performing scratch wound healing assay using HUVECs. I serum-starved my cells overnight prior to wounding my cells. I used EBM + all supplements except growth factors + 0.1%FBS as my starving media, as I found that the cells were not healthy (they start to lift and die) if I only use EBM without any supplement. I had BBE in my starve media.
Interestingly, 24 hours after human VEGFA stimulation, I did not manage to see an effect in the migration area of VEGF group compare to no VEGF control group. This is quite surprising considering VEGF is a potent angiogenic factor used in many HUVEC migrations studies (I used 25ng/ml, same as many other studies). Our VEGF and HUVEC batches are new and we used P3, cells looked healthy after 24 hours migration so the issue is unlikely to do with the cells. Could it be the starving condition? Can someone who has done this offer a suggestion?
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Hi All,
I just realised that I have completely forgotten to reply for almost a year! I did manage to optimize the protocol, and in the end my HUVEC got a pretty good response to VEGF (more than 150% migration 18 hours after treatment). I think the trick here is not to add BBE or any other supplements into the EBM starve media, other than FBS (0.5%), Glutamine and ascorbic acid. It is possible that these other supplements/growth factors may have other effects on the HUVECs (e.g proliferate) rather than migrate, which may be why I didn't see a response to VEGF. Also I did starvation for 4-6 hours only and not overnight.
Thanks for all your contributions! If you have an even better protocol, please do share.
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I observed growth inhibition/cell arrest coupled with cell migration at the same time during treatment of drug X that activates MAPK signaling.
Why are cancer cells arrested before they could migrate?
Are these cells preparing themselves (preparation phase?: attain enough signals, morphological alternation etc. ?) so that they could migrate easily?
Reference to your input will be highly appreciated.
#metastasis #Cancer #Hepatoma #signaling
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Tumors are very heterogeneous genetically, phenotypically and metabolically. With clonal evolution you have more than one kind of cells that evidently behave differently.
If all the cells would be equal, chemotherapy would solve all cancers. It does not.
In your case you have two different kind of populations.
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I was planning to perform young's moduli measurment of mammalian cells with atomic force microscopy (AFM). The live cells migrate when you try to perform AFM. I am thinking to fix the cells so that I don't have to worry about their migration. Does anyone know whether fixation would change the young's modulus of cells?
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After decomposition to formaldehyde in solution, it cross-links proteins inside the cell, making the cell much more rigid than living cells. See for example to work of Hoh and Schönenberger ().
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There is a bidirectional flow of fetal and maternal cells in pregnancy. Fetal cells migrate and resides in breast tissue, thyroid tissue, skin and so on in mothers body. How can we detect these fetal cells in mothers tissue?
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This is called microchimerism, sometimes -if sustained until few months postpartum- detectable by the immune system leading to autoimmune disease.
Detection methods include -beside Y chromosome as said above- HLA typing.
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I just read this paper
mentioning that
"Migration of cells in 3-D can be
expedited or minimized depending on the ratios of
matrix components such as collagens and glycosami-
noglycans."
referring to
but in the referred paper I didn't find further explanation of how is the cell migration is influenced by ECM composition
Could somebody offer a more detailed explanation on relation between ECM composition and cell migration?
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How to image analysis will help in understanding the molecular mechanisms that define cancer cell migration.
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You should perform "In vitro scratch assay" and do microscopy at several time points. Alternatively, you can also do Transwell cell migration assay and see the migrated cells under microscope after crystal violet staining.
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Recently, I want to use transwell assay to study glioma cells migration and invation. I have looked for a lot of protocols. And there were different opinions. I didn't find a complete and convenient protocol. What reagents and materials do I need to do this experiment? Thanks.
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Hi Shen Wu:
i totally understand what you are talking since i had similar experience several years ago. Quite a few candidate experiments suitable for this aim, such as transwell assay, invasion assay, and wound healing assay, every experiment has its cons and pros, and none of them is edge-cuttingly convincing. Since this work is critical for my project, i tried all 3 of them, the results generally consistent, so i drew a conclusion.
If this piece of work is not so sedimentary for your whole project, it would be a good idea to choose the one Charles recommended above.
Best regards
Shiqiu
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Hi guys,
I'm trying to do stats analysis on my scratch assay data in which I tested 4 different drugs, each at 5 different concentrations (along with negative control) on cell migration. I took a picture of the cells everyday for three days and analysed the cell numbers of each image on ImageJ, each with 4 repeats and an average found. I think I need to use 2-way ANOVA repeated measures to analyse any significant differences but am unsure of how to do this in minitab or SPSS and how the data is supposed to be laid out in minitab or SPSS?
Could anyone help me please? I would really appreciate it!
I've added my data here if it helps!
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Analysis of variance (ANOVA) can determine whether the means of three or more groups are different. ANOVA uses F-tests to statistically test the equality of means. In this article, I will show you how ANOVA and F-tests work by using a one-factor ANOVA example.
You can use the F test in the unidirectional ANOVA
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I am using Hela and SW480 cell lines. I need to incubate up to 24 hours -48 hours. How can I be sure that the wound closure in scratch assay is due to cell migration and not cell proliferation. I do not think that serum starvation is the best method for migration assay. also which dose of actinomycin D should I use ? Should I pre-treat the cells with Actinomycin D for only 2 hours (during the division cycle) before te scratch?
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Ons Zakraoui Hi, I am not a big fan of the scratch assay to quantitate cell migration. It is too subjective.
Scratch too light and you cant see the line. Scratch too hard and the cells have to cross a deep trench to get to the other side. I would use a transwell assay.
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I want to establish peritoneal macrophage migration assay by 8.0μm boyden chamber.Is it possible to compare the cell migration ability in different genotype of cells,for example WT cells and KO cells in response to chemokines. In this case,it would be 3 parameters changed. First,I think the cell number seeding in the chamber is not totally equal even if determining the cell number by cell counter.Second, cell genotype is different.Third,treatment is different (within or without chemokine in below well)
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I think that you have several other parameters that appear to be outside your control here. In the first place your macrophage population is not homogeneous, rather it is a mixture of maturities and state of activation. Heterogeneity will be even greater after manipulation/KO.
That said I still think that you may be able to get some useful data from your assay.
Please use a manual count, not a machine, to ensure your cell numbers are accurate!
Remember that your cells are populations! They are not clonal and not all equal!
The key to the Boyden chamber assay is timing. If you marginalise the assay (e.g. shorter times, serum-free, hypoxia) you will maximise your signal to noise ratio and hopefully observe a measurable difference between your cell populations.
How will you verify you have KO'd transcription in your KO cells?
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I have recently been trying to establish sub-cultures from human vascular smooth muscle cells via. LCM with limited success - i'm currently seeing approximately 60% of cut cells transferring across to the new plate still attached to the membrane, however the cells appear reluctant to migrate off the membrane & start dividing again. We are seeing some cells attaching to the plate surface however these look to be cells that have become detached from the membrane either during the catapulting process, or the transfer from the cap to the plate & this makes identification and tracking of individual cells difficult - does anyone have any experience in using LCM for this purpose? I'm new to this technique & have only found limited descriptions of the process in publications, any help would be greatly appreciated.
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With the LiveCell Chamber, MMI offers a dedicated system for live cell cutting:
Using this LiveCell Chamber, single or multiple cells from cell cultures can be isolated and recultivated easily and in a contamination-free way. Please feel free to contact me directly for any further information.
Kind regards,
Heidi
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I am a beginner in cell culture and I would like to understand why aren't these MSC in collagen type I gel migrating? I kept the gels in DMEM+FBS 10% at 37°C/5% CO2. These captures were taken 1 month apart. Cells were stained with DiI.
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Thank you!! I meant they are NOT showing any kind of displacement or movement, as I expected, but i don't know if maybe it is normal that there is no cell migration. I will explain the process:
I dissociate adherent cells from the flasks with trypsine, then I stained with DiI (5uL/10ml), I do this to stain them before seeding them in the hydrogel so I can "track them" when they are seeded. I let the DiI with the cells for 20 min in the incubator, then wash them with HBSS. I proceed to centrifugate and re-suspend in 1300 ul of collagen rat tail collagen type I EMD Millipore 08-115. Each scaffold or hydrogel was made with 300 uL cell suspension. Then I achieved collagen gelation using ammonia vapor chamber for 2-4 minutes. I only used 1% antibiotic once in the growth media because it was showing contamination but then stabilized. The hydrogels were kept in DME+10%FBS
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Dear all, recently i did a scratch test migration assay on my test compounds incubated with my cell line of interest and it appeared that the cell line seem to exhibit signs of increased cell migration. My question is, which proteins/genes related to cell migration/motility should i look at to further expand my current scope of research?
Thank you in advance for your kind responses.
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Dear Justin,
In the case of migration there's many things to look at that listing proteins would be not so helpful. Besides, many of them may have synergism with each other masking the target you are searching for.
Focus on your treatment, what it does. Gather info about the signal pathways it may be interfering with and from there infer possible candidates. Don't worry if you don't find immediate migration effectors in your search, they could be secondary downstream effectors associated with migration. Then you can start testing some of them.
Otherwise, by randomly pinpointing individual targets, you may end up wandering in the dark which surely will become more expensive and time consuming.
I don't know whether you are facing a short or long project, but if you are sure to have a migration phenotype and if you have the resources, I would recommend to do some proteomics on your control and treated samples. Then you can compare by protein ontology among several targets that changed expression pattern after
the treatment. This type of study also helps to better see the "full picture" of what your treatment causes to the cells.
Best,
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Does the electrode need to be of a specific metal?
Which salt solution and concentration to use?
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Interesting..
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I generally use GAPDH, alpha-tubulin, beta-tubulin and vinculin as loading control for my western blotting experiments and gapdh for qPCR analysis. But recent publications show that in the hematopoietic cells, the cells of my interest, metabolism, cell migration are subjected to changes at various levels during their different stages of development or differentiation, where the above molecules especially GAPDH and vinculin are widely known to be implicated. In light of these facts, I am confused whether to use or not the above mentioned proteins as loading controls to normalize the gene expression across various genetic alteration conditions. It would be very great of you, if you can share your view and suggest some way out of this confusion.
Thanks.
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Personal experience based on different gene manipulations (overexpression or knockdown) in mammalian cell culture, there is no single housekeeping gene.
For qPCR, better to make sure your input cDNA levels are the same and measure twice the RNA before reverse transcription. As housekeeping gene, you would give a try with Histone H3 and B2M also. whichever works best, you can go with that.
For Western Blot, you can also try Tubulin and Histone H3 in addition to yours. Also, I've seen that some papers have stained the membrane with Ponceau, took picture and put as control instead of a housekeeping protein. In this membrane, you would see the protein bands are pretty much similar. This was in a high journal as far I remember, either Science or Nature.
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Hi,
I'm working within a project on the role that hyaluronic acid (HA) in tumor cell migration and invasion. I have started testing low molecular weight oligosaccharides (maximum 18 repeat units), but these compounds seem to have little effect in our models. For this reason, I would like to try some type of higher molecular weight HA (HMWHA), but I'm not sure about which MW size(s) should I try. Also, I don't know how these HMWHA would be used in an in vitro cell migration/invasion system (soluble? gelified? wound healing or transwell?), so any help in these will be really appreciated.
Many thanks,
Jordi
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Jordi Senserrich - Hyaluronic acid for research use is available here and manufacured in Europe - Contipro ship all over the world
Take a look at the catalog attached on page 42 for laboratory grade - Sodium hyaluronate - the SH MW 1250 - 1500 kDa would suit the MW you are looking for.
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I don't understand the whole concept of epithelial-to-mesenchymal transition (EMT).
I believe it was proposed as an explanation of how circulating tumor epithelial cells migrate and colonize distal tissues. Normal epithelial cells do not migrate all that much.
But the EMT concept proposes that tumor epithelial cells differentiate into motile mesenchymal cells that migrate and then de-differentiate to the original tumor epithelial cells once they have colonized a tissue.
This could be plausible, but there are no positive markers for EMT. There is only subjective measurements of upregulation of THIS and downregulation of THAT.
Where is the proof that this EMT is real?
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Hi Steingrimur,
To give a better answer, it may be helpful if you would clarify what exactly you mean by "positive markers" and "subjective measurements of upregulation...". However, I will attempt to answer now and we can discuss our thoughts more once I think I truly understand your question.
First, the concept of EMT is not originally from the field of cancer, but rather from the wound healing process particularly cutaneous tissues, but the phenomena appears to much more broadly applicable. In this process, part of the final stages of wound resolution is the migration of healthy adjacent epithelia to recover and or close the wound site. In this setting, it was observed that, largely under the influence of TGF-beta (although this is admittedly highly oversimplified), the healthy epithelial cells undergo a series of changes in which they lose typical epithelial features including growth in a cohesive layer, apical-basal polarity, and polygonal shape to take on the characteristics of mesenchymal cells which allow them to better complete the healing process. These changes are accompanied by loss of expression of typical epithelial markers, notably E-cadherin, and gain in expression of N-cadherin (along with many others including the transcription factors that bring about these changes).
Additionally, when I think about EMT I do not really consider the process of EMT to be differentiation per se. I say this because the process of differentiation is thought to be a unidirectional process (That being said there is some evidence that terminally differentiated colonic epithelium has the ability to de-differentiate to regenerate the entire heterogeneous population of colonic epithelial cells including progenitors under conditions of extreme stress/ injury). Because of this EMT is a transient and reversible shift in phenotype driven by underlying cell signaling transcriptional programing. Thus the process seems more akin to the activation of a T-cell rather than the differentiation of a T-cell from a common lymphoid progenitor.
Because of this distinction, it may be better or more clear to say that cells that have undergone EMT are simply more mesenchymal in appearance and gene expression. That is to say that EMT is not purely binary, but rather a spectrum of epithelial-like to mesenchymal-like. If you consider EMT as a spectrum rather than two binary states, then it is easier to see why its hard to say specifically where a single cell is at on that spectrum if continuous levels of gene expression changes and continuous measure of cell migration are discounted. However, the problem is exacerbated by the fact that many of these studies use large populations of cells rather than single cells. Thus, there is a great deal of heterogeneity across the whole population at many points in the process of cells undergoing EMT (From ability to respond to the given stimulus to the arrangement/ modification of the chromatin containing the genes required to exhibit the mesenchymal phenotype and probably many more than the community is aware of). It is, however, interesting to consider that if EMT studies were carried out at the single cell level (perhaps through high quality flow staining or multifaceted gene reporter systems) then the process may indeed appear more binary allowing the generation of a more concrete definition of EMT at least under those experimental conditions.
As for the role of EMT in metastasis I will point you to a paper by Fisher KR from Vivek Mittal's group at Cornell (PMID: 26560033). In this paper they use a reporter system to longitudinally trace cells that had undergone EMT (at least one flavor of it). Using this set up, they show that cells that had successfully undergone the process of metastasis had for the most part not undergone an EMT. This is interesting work, but overall, I think that EMT has so many possible facets and molecules that can drive a cell further to the right on the epithelial mesenchymal spectrum that many more studies with a similar set up that look at different molecules will be required to demonstrate to what extent EMT is required for metastasis and how far to the right on the spectrum a cell needs to be before it acquires the ability to metastasize. Despite the shortcomings, I think that you may find the paper interesting given your skepticism of EMT and its role in cancer.
Finally, I will pose my own question for you or anyone else that is interested. In the paper mentioned above, they show that EMT confers resistance to chemotherapies. Additionally, it has been shown that cells that have under gone some extent of EMT divide slower. Finally, EMT is classically thought to be a major factor in the invasion metastasis cascade and adhesion independent growth/survival (Labelle M, Begum S, and Hynes RO PMID:22094253 among many more). All of these are key features of both EMT as well as cancer stem cells. My question is to what extent are cells that have undergone EMT and cancer stem cells different populations or are they actually highly overlapping cells populations or even in the most extreme interpretation different names for the same phenomenon (albeit with a different set of markers)? And if the later is true does the fact that the markers are different warrant a different name?
I hope this helps or at least inspires some thoughts and questions. Sorry for the book.
Best,
Andrew
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Hi everyone,
These day, I am doing cell invasion experiments using transwell inserts. I used collagen type I 1mg/ml.  The thing is, after invasion, I need cells move and attach to lower chamber surface, not the underside of membrane. However, as you know, in transwell assay, most of cells attach to the underside of membrane. 
So, can anyone help me which chemo attractant we can use for induce cell move and attach to lower chamber or any other  possible way in my case. Thanks
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Our company develops 3D cell barrier culture systems for in vitro study of virtually any cell barrier, and the system comes equipped with both a Fluid Perfusion Unit and a Trans-Endothelial/Epithelial Electrical Resistance (TEER) Measurement Unit! The use of a vessel-shaped 3D environment has been proven more effective than 2D systems like Transwell (
Article Santaguida S, Janigro D, Hossain M, Oby E, Rapp E, Cucullo L...
), and our advanced TEER Measurement Unit allows for frequency sweeps between 0.1-1,000 Hz, automated time point sampling, logging of data to Excel, and additional measurement of phase angle for cell capacitance calculations! For those that are committed to Transwell use, we also have a TEER Measurement Unit that is compatible with nearly all Transwell products (Endohm cell cup chamber, STX2 "chopstick" electrodes, etc) and allows the user to greatly expand their testing capabilities with Transwell equipment. Additionally, we have smaller modular systems that connect to microscope-friendly cell culture modules: i.e., a miniaturized 3D cell culture system that you can use right under your microscope! If you or anybody on this thread is interested in learning more, I encourage you to visit www.flocel.com or email me at djanigro@flocel.com, thank you!
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Dear all,
Recently i have a really crazy and brilliant idea to determine if a compound would be able to increase / decrease the motility of a cell type towards another cell type. How should i go about to prove this hypothesis?
Thank you for your kind responses.
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Depending on your throughput you can also look at the Dunn Chemotaxic chamber, or this higher throughput version from IncuCyte, which negates teh rollover effect you get with loss of gradient in a transwell.
You'll need access to an IncuCyte though
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I've been having trouble with this assay. In the control transwell chambers I see that the cells migrate uniformly, but in the ones with matrigel, only the cells in the edges invade (there is a circle without cells in the middle).
I'm using the Corning® BioCoat™ Matrigel® Invasion Chambers, so the problem is not due to the coating of the matrigel.
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Hi Sofia, I'm familiar with this issue.
I had a problem with uniformal cell migration when the meniscus in the upper chamber was close to the surface. The surface tension pushes the cells to the periphery.
The issue was solved by adding more media to the upper chamber so the meniscus does not come near the upper chamber surface.
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After 3 hours of incubation of cancer cells (in 150ul media 1%BSA) on top of the 8um pore size insert with known concentration of chemo-attractant at the bottom(1ml of media+chemoattractant 1%BSA), I see that the cells migrated more at the edges and less at the center. Since I need to count the cells at 3-4 different locations to quantify cell migration, this is a problem.
Can anyone suggest me how to solve this?
Thanks
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Hi Rahul,
I have tested the migration of murine and human melanoma cells by the modified Boyden Chamber under different concentrations of a tested agent, i think that you used the same concentration of the chemo-attractant (in 150ul media 1%BSA) in the top and bottom of the insert. there must be a difference in concentration like the bottom one is upper than that of the insert so that the cells can be attracted in this direction.
protocol
B16F1 melanoma invasiveness issued from resected tumour
was measured in vitro using Transwell® chambers (Sigma-Aldrich,
Saint Quentin, Fallavier, France) coated with Matrigel® (30 μg cm2,
BD Biosciences, France). Briefly, the in vitro invasion tests were
performed as follows: 50 000 tumour cells were suspended in
serum-free DMEM containing 0.2% bovine serum albumin (BSA)
and seeded on coated-membranes. DMEM supplemented with 2%
FBS and 2% BSA was used as a chemoattractant. After an
overnight incubation, cells were fixed with methanol and stained
with crystal violet. Cells remaining on the upper side of the
membranes were eliminated, and those on the lower side counted.
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I seeded LNCaP cells in transwell (medium: upper 1% FBS; lower 10% FBS). I found only few cells in the lower side of transwell after 48 hours. I performed the test with other cell lines obtaining good results.   
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@ Matthew A. Ingersoll, what percentage of FBS did you use in your wells.
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Hi:
We would like to stain the cancer cell cytoskeleton in cell migration and would like to know whether there is any difference if using fluorescent labeled mAbs (actin and tubulin) or small molecular specific binders (paclitaxel and phalloidin) and which one gives a better signal to noise ratio? Thanks.
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Phalloidin works much better than actin antibodies for fixed cell labeling. For microtubules, there is a monoclonal antibody (clone DM1A) that works great.
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Can cell migration/invasion in the presence and absence of pharmacological inhibitors be tested for 1 week using this assay?
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The duration of experiment may differ depending on the cancer cell line you use, also the method and conditions (normoxia/hypoxia).
There are some different assays to test cell invasion/migration. Some of them may be done in 5-7 days (inverse invasion assay), some in 8-10 days (3D invadopodia formation), etc.
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Hello everybody,
I am trying to use the ibidi chamber to perform migration assay with glioblastoma cells (U87-MG) and I add 0.5% of FBS in one chamber as chemoactant. I've got the timelapse and cells do not seem to migrate at all. May be 0.5% FBS too low?
Thank you much!
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Hello Daniele.
Good luck.
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Hello. At the start of my video I have a different cell density of control versus treated (protein silenced) cells. I wonder if this difference in cell density would affect the random cell migration?
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Dear Stephanie,
there are are a number of physical (e.g. cell collisions, cell-cell adhesions, etc) and chemical (e.g. secretion of chemotactic and growth factors, matrix deposition, etc.) produced by cells that can influence the (migratory) behavior of their neighbors.
Thus, cell density is essential for the correct execution and interpretation of the assay.
Best,
Metello
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a lot of data show that CXCL12 can promoter cancer cell migration
experiment process
1. coating matricgel(1:9 in seriumfreeDMEM) transwell-chamber (SPLinsert 8 um) put into the Cell Culture Incubator 6hours
2. count the cell 2*105 use the non-seruim DMEM wash twice
3. inside chamber add A549 cell combine CXCL12 cytokine (100 ng/mL)
outside chamber add 10% DMEM
4. put in bake Cell Culture Incubator 14 hours than fixing and staining
this is my problem "CXCL12 treatment migrated cell is lower than the control"
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Hi, if CXCL12 promotes cell migration, you have to put it outside the cell chamber. The cells have to migrate towards a stimuli.
If you put the stimulus in with the cells, they have nothing to migrate towards.
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Both cell proliferation and migration contribute to reepithelization of wounded skin. To observe the effects of any genes or compounds on cell migration, we usually use the scratch assay to assess the keratinocyte motility, and regard it as a measure for the cell migration in vivo. Normally, in the scratch assay we treat the cells with mitomycin c to block the cells' proliferation. Can we treat mice with any small molecular inhibitors to block the proliferation of keratinocytes so that we may assess the cell migration in vivo? If this is not possible, are there any other methods that can be applied for this purpose?
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I found an answer to the question. Dr. Ce´dric Blanpain lab applied 5-fluorouracil (5-FU) to block epidermal cell proliferation. Please refer to: DOI: 10.1038/ncomms14684
,
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hi everyone,
I have two questions-
1. Can we knock out the mouse gene in both cell- and tissue- specific manner?
2. If so, can we further get the mice whose CD4+ cells in spleen only are lack of the gene? Is this impossible due to the migration of wild-type CD4+ cells into spleen? Or aforementioned migration will never happen though the T cells will migrate out of secondary lymphoid organs once differentiated sufficiently.
Thanks, and any comments are welcome and appreciated.
Jiyuan
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The situation you described is conditional gene knock out or knock in.
The main strategy is Cre/Loxp system. It can control special gene express or not at certain time or condition.
Hope it will help.
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Hi all,
During the in vitro metabolism of fibroblasts, what kind of waste this cell generate? We are trying to explain how these wastes may affect the cell migration.
Thanks a lot for your suggestion,
Long.
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Hi all,
Is there any study on the relationship between immune cells migration and immune surveillance, or immune escaping of tumor cells? It is clear that immune cells migrate in different tissues and this migration helps them to complete their functions. One of these functions, immune surveillance, should be under effect of immune cells migration but I can not find anything. I am looking for related papers or a keyword.
Thanks
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Here are 2 reviews on homing of immune cells/T cells to solid cancers:
Ager A et al. Homing to solid cancers: a vascular checkpoint in adoptive cell therapy using CAR T-cells. Biochem Soc Trans 2016;44:377-85
Tan LY et al. control of immune cell entry through the tumour vasculature: a missing link in optimising melanoma therapy? Clin Transl Immunol 2017;6:e134
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My cells were transfected with Wnt5A containing vector and stably express Wnt5A. In scratch assay, these cells show increased migration relative to mock but in transwell migration assay ,their migration and invasion is lower than mock cells.Is there a specific reason for this?
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I agree with Vilma. Your cells with enhanced Wnt5A might only have effects on migration but not invasion. Compared to in vitro cell culture, you can use mice to see the in vivo effects of increased Wnt5A. After all, the differences between them are huge.
Besides, you can also detect some pathways related to migration and invasion, i.e., FAK pathway. Some chemokines can also be measured as well.
Good luck!
Jennifer
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We are trying the Boyden chamber without results. Our problem is on cell visualization in the membrane.
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Dear Pedro,
I agree with Teresa. I used the Boyden chamber (and relative modification, such as the transwell insert) a lot and I never really liked it. The results are quite variable and the visualisation system not that reliable.
I personally never used the Ibidi chambers, but I use to make something similar connecting two 3.5 dishes with a glass capillar.
Another good alternative especially if you do not need high magnification, is the Dunn chamber; in this case the migration is from the center to the periphery so it is also relative easy to quantify the behaviour of a cell population. The down side is that if you need to go higher than 40x and use oil it become un-pratical.
Hope this help.
Good Luck!
Max
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Hello,
I'm very much aware of the difference between cell migration in 2D vs 3D. As many papers and PIs have always mentioned this. However, just how difference this is, i dont really have an idea. 
So to make this more specific: There are two cell lines that I have got: 1 stably expressing a mutated protein of interest while the other cell line only contains the empty vector. When I did random migration assay, which literally just seed the cells on gelatin coated 6-well plate and do a timelapse movie of them, the mutated cell line unexpectedly display a very random undirectional movement, while the one with the empty vector display a more directional movement. This is similar to cell line stably expressing the wild-type version of the protein. 
However, a previous experiment done by someone else in the lab found out that the cell line expressing the mutated protein invade deeper into the matrigel (they did an inverted invasion assay). And this doesn't seem to add up with what I saw on 2D.
So there must be such difference between 2D and 3D, but is it possible to have a complete opposite effect? 
Any opinion would be appreciated! 
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Hi Anh, being scared, nervous and doubting yourself is an occupational hazard of being a scientist.
You are not the first or the last scientist to doubt what you are doing will be of any importance.
Believe me, I have had my share of nail-biting doubts that kept me awake for days on end and I failed many times in my experiments.
I can't explain it, but it is like science chose me rather than I chose science . I don't know why that is.
But please remember, you are not alone.
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I am working on detection of some proteins secreted from cell associated with cell migrations and I try to notice migration of these cancer cells but they don't migrate. What I should add to these cells to make them to migrate.
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Hello, you could use a chemokine such as CCL2 or CXCL12 to induce migration via the CCR2 and CXC4 pathway, respectively.
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Does anyone have experience with migration or invasiveness assays in A549 cells in 75mm transwell?
Since 75 mm transwell is pro ducted only in 0.4 or 3.0 um, is 3um pore size suitable for this cells?
Thanks :)
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Thank you all for your answers! I found that also 5 micron transwells work for these cells. I should try with 3 um. 
Agnieszka, no treatment with drugs. I cannot use wound healing assay instead, because I need to recover migrated cells. 
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I have seen in literature, people have shown figures of transwell for migration and invasion assay (suspension cells) but as far as i know, when we will try to fix the suspension cells they will leave the membrane of transwell. Counting the cells is another method but if there is any possibility to show figures of transwell ....please suggest the protocol.
Thanks
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I quantified the suspension cells in transwell assay by the addition of calcein AM (4 μg/ml) to the lower chamber for 1 hour as described by the manufacturer (Biotium) and total fluorescence measured using a Synergy HT plate reader (Biotek).
Or stained the target cells from mix of primary tissue after invasion or migration assay by fluorochrome conjugated Abs. (https://www.ncbi.nlm.nih.gov/pubmed/24091323)
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Hello,
I am working with cell suspension derived protoplasts of celery (Apium graveolens) and sometimes spontaneous agglutination is occuring during the isolation, while the cells are suspended in W5. I observed that this phenomenon is not always occuring and I think it would depend on the cell line. Since this is not desired for my experiments, I was wondering if there is knowledge about the cause of protoplasts agglutination (except pathogens elicitors, the culture are sterile) and how could it be prevented.
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I come here with some updates on my work. Decreasing Ca in the bufferes helps against agglutination. I tried resting the cells at 8-10°C or even lower and that helps too. Indeed when the lysis process starts, it expands fast and cells died in a matter of minutes.
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I studying a effect of 'A' gene on angiogenic properties using HUVECs.
When Knock-down for 'A' gene, wound healing ability significantly decreased.
However, when knock-down for 'A' gene,  tube formation ability increased.
I can understand the different results of the wound healing (cell migration) and tube formation.
Any similar results has been reported? 
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did you coat the wells with gelatin for migration( woung healing) and with matrigel for tube formation?
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I am currently researching staining techniques for corning collagen coated transwell inserts (6.5mm). Does anyone know of any protocol that outlines how to stain inserts with H&E? Any information would be appreciated! 
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This is the method I use when staining the inserts:
1. Using a cotton bud, wipe any un-invaded cells from the top of the membrane.
2. Place the insert into 100% ethanol to fix for 5 minutes.
3.Stain the membrane with 1% eosin for 1 minute, wash in tap water, then stain with haematoxylin solution for 5 minutes and wash in tap water.
4. Carefully remove the membrane from the insert using a scalpel blade and mount onto a glass slide using an aqueous mounting fluid.
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i grew cancer cells on a transwell insert and then place an agarose cushion over the cells, followed by a weight on top of it. i notice that if the cells are more confluent (100 percent for instance), they survive the compression better. However, if they are about 60-80 percent confluent, they die.
Any reasonings for this.?? is force distributed laterally or do confluent cells have more tight junctions, protein-protein interactions allowing better coping of stress?
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Notably, confluent cell layers are more robust against any kind of stress, mechanical and chemical.
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Scratch wound assay are commonly used to measure cancer cell migration in response to a treatment. How to be sure that the data reflect the actual cell migration and not cell proliferation?
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Hi Aurélie, You need to add Cytosine β-d-arabinofuranoside hydrochloride (Sigma) (10 μM), a selective inhibitor of DNA synthesis, which does not inhibit RNA synthesis. 
You can find a protocol in this paper: https://www.ncbi.nlm.nih.gov/pubmed/24904530. Good luck
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I'm trying to do an invasion assay using glioma stem cells and I'm trying to determine my options. I found that two common methods of testing invasion are the matrigel invasion assay and the Boyden chamber. However, when I look at those methods, they seem pretty similar. They both seem to be looking at how cells invade (either a matrigel or a porous membrane) toward a chemoattractive substance. Can anyone tell me what the difference is between them and if there any other assays I should consider? Thanks!
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Hi Namratha 
For invasion I use matrigel invasion assay. The matrigel serves as a reconstituted basement membrane. Invasive cells will degrade the matrix and on doing so "invade".
On the other hand the Boyden chamber (just the porous membrane) is normally used for migration assays. As you mentioned the cells will move towards a chemoattractive substance. They don't really degrade any matrix, just move through the porous membrane.
Hope this helps,
Good luck
Silvia
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I have some very transparent small agarose heparin beads that I want to bind FGFs to then deviate cell migrations in chicken embryos with the growth factor. I would like to stain the beads but retain the colour and FGF binding ability of the beads so that I can see them when I place them on the embryo. Any ideas of stains that would not be toxic to embryos?
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I also electroporate GFP cells to trace their normal or abnormal migrations but it is specifically the bead that I want to stain so that I can see where I place it and see it the next day. Something like fast green that would retain the bead colour but not be toxic? Did you do this at all?
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Why the Endothelial Cell Migrate under the effect of DMSO faster than the endothelial cells moving in normal medium?
In my experiment i tried to know the effect of DMSO on Endothelial Cell Migration, so the results came with higher movementsof cells under the effect of DMSO more than the movement of untreated cells, do you have explaination related to that?
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You most welcome
Houda
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What is the best programme to track the cells migration and how it works?
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Get ImageJ; or Java version ScionImage; or Fiji (ImageJ v2).
All are freeware and can be used with most image file formats.
DIL
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Hi guys,
I recently developed a HEK293 cell line with an expression reporter at the endogenous locus of a gene interacting with EMT-associated genes. To test the functionality of the reporter based on such interactions, we want to induce EMT in these cells with TGF-Beta1. However, I've looked very hard for previous literature describing EMT in HEK293 cells, specifically induced by TGF-Beta, preferably with morphological data, but I cannot find anything. Does anyone know a good source to address my question? I'm concerned the lack of literature suggests this isn't easy to do.
Thanks!
Kevin
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Hi guys, this is an interesting point, we have the same issue. I wonder if HEK293 would go to MET, the opposite, as they express N-cadherin? 
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I want to figure out what technique biologists use to visualize cell migration and how do they analyze that.
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2 photon microscopy, Multiphoton microscopy imagine is the coolest way to visualise invivo cell migration, with the many florescent tag available out there, you can looks at how different cells migrate in response to stimulus.  But is every expensive assay :0 
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I verified expression of my protein of interest in parental and shscr THP-1s is the same. Then, I did a migration assay on Transwell chambers and I got a night and day difference in the number of migrated cells, with the knock-down cell numbers falling in between parental and shscr. I would appreciate reading comments from anyone who has experienced something similar.
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Maria,
   In some collaborative work that my lab has been involved in, we have seen that lentivirus itself can affect the behavior of transfected cells, including migratory behaviors. We also have seen some pretty spectacular examples of what looks to be cell fusion following lentivirus treatment-- Cells appear to fuse together and become one big, ugly  multinucleate cell that looks like someone took a dozen eggs and fried them all together sunny-side up. The cytoskeletal organization in these monsters is all screwed up, too. It looks like the stress fibers from individual cells are maintained as they were when fusion occurred, but the microtubules lose their normal organization and form huge networks thattend to push all the way out to the cell margins.
    I would recommend very carefully titrating your lentivirus dosing, and confirming your knockdown (or overexpression of a transgene) for each experiment.
Dave Sherry
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Dear colleagues,
I would like to do a Transwell cell migration assay, staining migrated cells by crystal violet and counting cells by light microscope. Unfortunately, I meet two problems in my experiment.
One was that I couldn’t distribute cells evenly in Transwell inserts, cells to either congregate in the center or along the outside of the wells. Therefore, for the same Transwell insert, the fields under microscope were not uniform, and some field was full of cells and some field had little cells, resulting in big error in cells counting. In experiment, I diluted cell suspension to necessary seeding concentration in tubes and mixed the cell suspension repeatedly, then added the suspension to the insert. And I tried shaking plates backward and forward, then right to left to right after seeding, or not shaking at all, but the problem couldn’t be solved.
Another problem was that cells weren’t clear under microscope after crystal violet staining (the color was very light), so it’s hard to count cells. I tried extending staining time from 0.5h to 1.5h but it didn’t help. Should I shake the plate or increase temperature to 37 degree? Or should the membrane of insert be dry before staining? And any other suggestion or tips for crystal violet staining?
The experiments details were as follows:
Cell: Hela cell
Transwell: Corning Transwell cell culture inserts, TC treated, PET membrane, diam. 6.5 mm, pore size 8.0 μm
Seeding volume: 50,000-100,000 cells in 0.1 ml serum free DMEM per well
Reservoir Volume: 0.6 ml 10% FBS in DMEM per well
Migration time: 16-20 h
Crystal violet: 500ul solution (0.1g crystal violet in 10 ml methol) per well
Thank you in advance for your suggestions!
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Hi Jiahui.
Let me tell you what has worked for me in migration assays. I have used inserts like yours for 24 well plates. The migration has always been done with 1ml total volume, 0.5 ml on top (cells) and 0.5ml in the bottom (chemoattractant). Let's start at the bottom, usually I have used anywhere from 0.1 to 1% FBS as chemoattractant, be aware that whatever value you use you must double the value because you have 1ml total volume and only 0.5 ml go to the bottom. So if you want 1% chemoattractant, you put 0.5ml of media at 2% FBS in the bottom well. For the top well the cells go in 0.5ml of serum free media. This allows for equal distribution of cells. The amount of cells you have to titer to the cell line used, usually anywhere from 10000 to 50000 per well. Some cells produce extracellular matrix and when there are a lot of them in a small volume they quickly clump and go uneven onto the surface. That's why you have to titer the amount of cells to give you a good result. Once you set your migration don't disturb it let it go for 16-24hr.
Now for the staining I have used Diff Quick stain (available from fisher and other companies that sell hematologic stains) This is a modified Wrights Giemsa stain it comes with 3 bottles (light blue, red and purple) The light blue is a fixative, red and purple are stains. So after migration I clean the inside of the insert with a cotton swab to remove non migrated cells. I place 1ml or so of the three solutions in 24 well plate and place the cleaned insert in the blue fixative, after 1 min then I place it upside down on paper towel to remove excess fixative and transfer to the red well for 1 min, repeat towel step to remove red dye and place in purple for 1 min, towel again and then dip the insert in a big container with water (tap is fine) and finally place upside down again to let dry. I use forceps to grab the inserts by their flaps, also I reuse the solutions and place several wells with solutions and do several 3 or 4 at the time. The timing of 1 min per stain is approximate, if it takes longer don't worry you cannot overstain.
Once the stained filters have dried I cut them out of the insert (use a fine scalpel) and I mount them on glass slides with mounting media and coverslips. later you can look at them at the microscope and count them, Also you have a permanent slide of the experiment that you can keep for recount, take pics etc. This method has always worked for me. I tried using crystal violet once and it was  messy and inconsistent.
I hope this helps you,
good luck,
edgardo
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Dear all,
I have been trying to optimize a transwell migration assay. However I have some difficulties regarding the counting part. I am doing crstal violet staining and  cleaning the upper part of the membrane with a kimtech paper. (Because the surface of 96 well transwell membrane is too small for regular cotton swabs) Then I cut the transwell membrane, mount them and later I clean the upper part again and take some pictures. But I don't know which cells I should count.
Here I attached a picture that I took in 63X and these are my questions:
-Number 1, 2 and 3 are the pores?
-Number 4 is a cell which is about to migrate through the pore?
-Number 5 is the right cell to count for the assay?
I would be really glad if you can give me some feedbacks. 
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Your criteria for designating a cell as 'migrated' may need to be more stringent, but as long as you count all conditions with the same threshhold you should see a phenotype. 
1. Many people only count cells that spread out after crawling through the membrane. The cells in the image appear to be stuck in the pores. Adjusting time of assay (at least 6 hours)  or serum/chemoattractant in the bottom chamber can help solve that problem. The speed of migration will be cell type/context dependent. 
2. At 63x , the residual cells on the top (unmigrated), are usually out of focus and will not be included in the count.
3. For washing the cells from the top, use a PBS-soaked cotton swab and a twisting motion to remove nonmigrated cells. 
4. Alternatively, you can trypsinize the membranes and the migrated cells will detach and fall/adhere to the bottom plate.
Good luck!
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Primordial germ cells show several features of motile cells. However some authors suggested that the migration due to morphogenetic movements and local cell divisions. Do these cells migrate actively or passively?I would like to share ideas about this subject. 
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Try adding 5micromolar Amlodipine to the flask. If you see a reduced motility the motility is actin dependent and is active.
Best,
Mannan 
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I am on a trial to demonstrate the cell migration under knockdown conditions. Is anyone experienced with such assay for murine cardiomyocytes.HL1 (adult). Any suggestion will be highly appreciated. Thanks in advance 
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Thank you so much, I will follow your ideas. 
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Initially, our cell migration experiments were going smoothly, with RAW 264.7 cells (grown in DMEM with 10% FBS and plated in serum-free media) responding appropriately to MCP-1. 
Then the cells stopped migrating in response to MCP-1. We were able to remedy this by adding 1% FBS to the cells at plating.
Now, the cells are migrating equally in the presence or absence of MCP-1.
This is regardless of whether the cells are plated in serum-free DMEM/RPMI or with 0.1% FBS/BSA. We have not been serum starving the cells in advance.
The cells will migrate when the bottom chamber only contains media with 0.1% FBS/BSA, even when they are also plated in media with 0.1% FBS/BSA. If MCP-1 is added, the migration is equally robust.
Any ideas as to why our cells migrate no matter what the conditions would be appreciated! Thank you!
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NP, Maureen! Good Luck with your following troubleshooting, will discuss it after.
"We have not tried a condition in which there is serum in the top, but not in the bottom, but we will try that tonight. "
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working on a cell migration study wherein I'm planning to study the migration of cells when placed in a double gel environment. I wanna know if we can prepare a gel system with two different colors so that when imaged we can find the cellular movement.
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Thanks Mr.Soheil ...
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Hi,
I'm planning to do a migration experiment where A549 cells are migrating into a Matrigel or similar. Most protocols use microscopy as a read-out method, but I wonder if it's possible to melt the gel again at 4°C to release the migrated cells and thereafter do proper cell counting in e.g. FACS? Of course, then the gel has to be properly washed away to avoid clogging in the FACS. Is it doable, does anyone have experience with similar set ups? 
Annette
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Hi Annette, I think the IncuCyte machine of Essen Bioscience can help you. We just bought one. Best regards, Evelien
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In particular, I'm interested in the time course of infiltration/differentiation in response to peripheral immune stimulus, such as intraperitoneal lipopolysaccharide injection. Thanks!
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Hi Smith
Look these links PLZ
Functional differences between microglia and monocytes after ...
by RM Ritzel - ‎2015 - ‎Cited by 18 - ‎Related articles
Given the high degree of macrophage heterogeneity that comprises our ... differences between brain-resident microglia and infiltrating monocytes in acute ..... This parallels the time course of leukocyte infiltration into the brain, which also ...
Functional differences between microglia and monocytes after ... - NCBI
National Center for Biotechnology Information
by RM Ritzel - ‎2015 - ‎Cited by 18 - ‎Related articles
May 29, 2015 - Functional differences between microglia and monocytes after ischemic stroke. ... to stroke is primarily mediated by microglia, the resident macrophage of the CNS. ... blood-brain barrier is compromised and monocyte infiltration occurs. ... function of each cell type following stroke over the course of 7 days.
Microglia, macrophages, perivascular macrophages, and pericytes: a ...
Journal of Leukocyte Biology
by GJ Guillemin - ‎2004 - ‎Cited by 409 - ‎Related articles
Nov 11, 2003 - Once in the brain, monocytes differentiate into one of the four types of brain .... BBB is altered and/or leukocytic infiltration in the brain parenchyma, most of ... Changes in the cell morphology are, of course, very significant [ , , ,].
Differential roles of microglia and monocytes in the inflamed central ...
jem.rupress.org › 2014 Archive › 28 July
Journal of Experimental Medicine
by R Yamasaki - ‎2014 - ‎Cited by 128 - ‎Related articles
Jul 7, 2014 - Macrophages may be derived from infiltrating monocytes or resident microglia, .... MDMs and MiDMs exhibit different time courses of accumulation in the ... Morphological features distinguish MDMs and MiDMs at EAE onset ..... with infiltrating monocytes or neuroepithelial brain cells (Butovsky et al., 2014).
Frontiers | Microglia and monocyte-derived macrophages: functionally ...
by A London - ‎2013 - ‎Cited by 76 - ‎Related articles
Our studies have led us to view the myeloid-derived infiltrating cells as functionally ... microglia, and accordingly, to name them monocyte-derived macrophages (mo-MΦ). ... It allowed the study of non-activated microglia in intact brains of living ... direct the migration and differentiation of NPCs, as well as secreting soluble ...
Infiltration Pattern of Blood Monocytes into the Central Nervous ...
by R Menasria - ‎2015 - ‎Related articles
Dec 23, 2015 - To distinguish microglia from blood monocyte-derived macrophages, ... into the brain of infected mice followed different time courses with a ...
Cerebral Microglia Recruit Monocytes into the Brain in Response to ...
The Journal of Neuroscience
by C D'Mello - ‎2009 - ‎Cited by 245 - ‎Related articles
Feb 18, 2009 - Furthermore, in mice with hepatic inflammation, microglia were activated .... to be able to distinguish between resident microglia and CNS infiltrating monocytes. ... recognizes both microglia and macrophages (Ahmed et al., 2007). ... To confirm the inhibition of cerebral monocyte infiltration, BDR mice were ...
Prof. Dr. Awatif H. Issa
best regards
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Hi, guys, I know it's not easy to culture primary cells because they have limited cell division.
But for us, we are only able to passage cells once or even worse.
1) Cells are either growing slowly or enter senescence easily.
2) After cell passaging, the morphology also changes .
We are culturing ovarian cancer cells, and use DMEM+EGF+FGF+B27 with 0~2% FBS.
I also attached some pictures here from your reference.
Does anyone  know what happen? and how to modify the culture condition to make the cells in proliferation and also maintain the morphology?
Thanks a lot!!
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Hi Jingwen - this is a very difficult question to answer.
Some cancers, whilst being very aggressive in the human, just do not thrive in 2D culture conditions (triple-negative breast cancers is one example, there are only a handful of these cell lines available because of this). Therefore it might just not be possible to establish a cell line from your primary cancer (so may not be anything that you are doing wrong).
Senescence can be triggered by a number of factors. Mainly it is caused  by telomere erosion but it can also occur due t "culture shock" (i.e. growth on plastic). However, saying that, you might want to check for telomerase activity in the cancer cells (if you can) just to verify that it is active.
For some primary cells growth in high glucose media can  hasten senescent onset - so  check which DMEM you are using (and if high glucose try a low glucose one).
You may also want to add some Insulin to you media (some ovarian cell lines require this).
You could also try increasing the FBS (i.e. 5-10%) with a portion of your cells to see if this helps.
When making the initial splits try to keep the density of the splits fairly high i.e. 1 in 2 split rather than a 1:4. This increases the labour but some cells are density dependent and die when they get lonely. Initially high splits may help them survive until they can adapt to the culture conditions
A last suggestion: (and this is "grasping at straws" a bit) save the media from the cells before you split them and when you seed the new flasks feed the cells with  a 50:50 mix of old media (from the cells pre-split) and fresh media. (this is often called "conditioned media" in papers and protocols.).
One last thing - primary cancers are likely to be very heterogeneous (containing cells with numerous different DNA mutations) and 2D culture conditions will always cause a "selective pressure" (so faster growing cell or cells that can tolerate growth on plastic will come to dominate the culture over time). Because of this you will always get some "phenotypic drift" and hence morphological changes over time (so make a master bank of cells early).
Sorry I can't be more help!
Gary
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Hi everyone,
I would like to do a cell scratch experiment to check cell migration. Later I will use the migration area for the result analysis. Besides this, the migration border length should be measured as well. Does anyone know any softwares, which is able to measure the border length? (see the red line on the picture)
Thanks a lot!
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