Science topic

Endothelial Cells - Science topic

Highly specialized EPITHELIAL CELLS that line the HEART; BLOOD VESSELS; and lymph vessels, forming the ENDOTHELIUM. They are polygonal in shape and joined together by TIGHT JUNCTIONS. The tight junctions allow for variable permeability to specific macromolecules that are transported across the endothelial layer.
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I am wondering if a (semi) solid substrate/scaffold (could be a hydrogel) is necessary for endothelial cells to sprout out to a VEGF producing source. In other words, it it possible to have vascularization in a culture dish without any specific scaffold?
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Angiogenesis is a complex process in which the growth of normal, stable, and functional vessels is critically dependent on the coordinated interplay in space and time of different cell types and growth factors.
Binding of VEGF to ECM is critical for proper vascular morphogenesis. The formation of microenvironmental VEGF gradients in the tissue matrix guides new capillary sprouting. The first endothelial cells activated by VEGF become specialized tip cells, which sense the VEGF gradient by extending thin filopodial processes and migrate toward its source.
Studies have shown that appropriate internal scaffold structure can allow the nutrients to infiltrate and provide pathways for new blood vessel ingrowth. The pore size and interconnections between macropores are porous scaffold structure parameters and are two essential factors for blood vessel growth. Although pore size has a strong impact on vascularization, pore interconnection is more critical.
The spatial localization of angiogenic signals in the extracellular matrix is crucial to ensure the proper assembly and maturation of new vascular structures. So, ECM is necessary for vascularization.
Best.
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We want to check the inhibition effect of aflibercept on Huvec cell line.
In this way, we purchased a commercial HUVEC cell line and checked the dependency of this cell line on VEGF-A. HUVEC cell line grows well in complete media containing DMEM-F12 and FBS 10% (without any growth factor like VEGF-A), while we considered that proliferation of HUVEC cell line was reduced in the absence of a growth factor (VEGF-A).
we reduced the concentration of FBS to 2.5% to see the effect of VEGF-A on HUVEC cell line proliferation, but HUVEC cell line still grows independent of various concentrations of VEGF-A (20, 200 and 2000 ng/ml) with Calcein AM.
The Scratch assay was done and no effect was seen after VEGF incubation. Cells were incubated for overnight in serum-free media and then cultured with DMEM-F12 media, 2.5% FBS and various concentrations of VEGF-A (20, 200 and 2000 ng/ml).
whether huvec cell line loses this feature over multiple passages? or did we miss something? How we could check the function of VEGF-A? In this situation, how we can evaluate the effect of Afliberecpt on HUVEC cell line?
I would be grateful if you could help us in this matter.
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FBS allready contain plenty of not so cell specific growth factors, different kinds of endothelium have different growth requirements, HUVECS are primary cells obtained from vein of somebody-s umbilical cord, short culture only is recomended (7-10 passages?)
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I am assuming that endocrine cells should have no to minimum distance to the blood stream, so that the secreted hormone could be directly secreted to the blood, without passing through the ECM.
This brings me to the real question: Are pancreatic beta cells mechanically influenced by the intra-islet endothelial cells?
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Good evening,
I have started my culture with HUVEC cells and HAoEC, I use Endothelial Cell Medium
(Cat1001) from sciencell, so the complete medium contains FBS, antibiotics and endothelial cell growth supplement (ECGS). I thawed HUVEC and seeded in T25 without fibronectine and I used fibronectine to cover the bottom of the T25 for HAoEC. Both cell types don't want to grow properlly, there are a lot of dead cells in the medium and very little, round shaped attached to the bottle. Any advices? I would be very grateful.
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Hi Anna,
From your description I assume the problem is in cell thawing. If your ECs do not adhere to fibronectin-coated dish(es) it suggests that they are inured/dead :(
If the "classical" cell thawing/seeding procedure is not working for you, consider the following to minimize cell death/loss :
1) Thaw your frozen EC without "shaking". Don't keep the vial in the water bath (+37oC) for too long (just long enough to dissolve "ice crystals").
2) Once the" ice crystals" in the vial will be gone, gently suspend/transfer the cells in to the tube with 10-20 ml of complete medium (depending on the cell culture dish you are planning to use). At this point it is important to dilute DMSO (present in the cell freezing medium) as much as possible.
3) DO NOT centrifuge!
4) Transfer the whole content of the tube (10-20ml) in to T25/T75 dish. Allow cells to adhere for 2-3 hrs. This period of time is long enough for EC to adhere and spread on Fibronectin-coated surfaces (in most cases 1 hr is long enough for cells to adhere). You can check cell adhesion under phase contrast microscope.
5) Once the cells are attached, collect the medium with remaining not attached cells into a separate tube, and place new fresh EC growth medium to the attached cells. The attached cells should be happy :)
6) At this point you may centrifuge the collected medium (with remaining not attached cells) to remove DMSO and plate them in to a new T25. With this step you will maximize recovery of your original cell stock.
Best of luck!
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I see it added to some formulations, but when I search around online for what it does I don't see clear answers! I also see conflicting information on whether it's good or bad for endothelial cells.
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Thank you Maria Cinta Cid Xutgla and Malcolm Nobre ! I also just came across this article that looked at how heparin helps ensure wild-type FGF2 stability
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I am about to begin transmission electron micrographic studies on the Brain cortex NVU in mice models infused with lipopolysaccharide (LPS). Initially, I will investigate to see if there is a loss of the brain endothelial cell glycocalyx and then also study the mural cell pericyte and the neuroglia. It is known that Microglia and Astrocytes are activated by LPS; however, it is not known if there is activation of the brain endothelial cells to result in increased transcytotic vesicles and thus increased brain endothelial cell permeability without affecting the BBB tight and adherens junctions.
I look forward to any and all input from this question. I am hungary to know what you all think regarding this new experiment. I really hope to learn from this question and your answers.
I will begin once the omicron variant settles down as it has been raging here in central US now for over 2 weeks and creating a huge stress on our hospitals and health care providers.
Please feel free to share your opinions and or some of your experimental findings from publications and your knowledge.
Sincerely,
Melvin Hayden
University of Missouri School of Medicine
Columbia, Missouri USA
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Next week I will finally be able to obtain TEM from the LPS infused models and will post some of my new findings once I have acquired them to share.
If you have any ideas or suggestions please share. I will be examining the endothelial glycocalyx with lanthanum nitrate staining and examining for endothelial cell increased vesiculation and activated microglia cells and also examining the Astrocytes to see if there is any remodeling to a reactive phenotype .
I look forward to reading any and all of your comments regarding the study of LPS infused mice models and future TEM ultrastructural remodeling.
Melvin R Hayden
University of Missouri School of Medicine
Columbia, Missouri USA
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Hi all,
We're having problems growing primary human liver-derived endothelial cell lines (purchased from Lonza), and wondered whether anyone has had success growing these cells/ would be wiling to share their protocol?
The first lot of cells looked great at first recovery, but never went beyond passage 1 as they just stopped growing.
The second lot of cells unfortunately never even attached to the T25 flask after being thawed!
We've successfully worked with a wide variety of endothelial cell lines in the past, so I don't think our cell culture technique is the problem.
Would be grateful for any thoughts/ suggestions.
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We faced same problem with Lonza's media.
When we changed provided FBS to another company's FBS(maybe corning's), We can culture healthy cells.
We are using another Endothelial medium now.
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Hello,
I cultured two plates of endothelial cells using different media formulations (control vs. experimental). I want to see if levels of an intracellular protein have been altered in the experimental condition. Western blotting or adherent-cell immunofluorescence both seem like attractive options to do this.
Immunofluorescence is beautiful - but is it a more efficacious method than western blotting?
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You need to choose a technique to match your end goal.
I would recommend you go for Western Blot which is a reliable semi-quantitative method only if sample properties and integrity, antibody specificity to the target protein, and proper loading protocols (loading conditions and loading controls) are considered. Semi-quantitative because you will be determining whether the expression of the target protein in the experimental has increased or decreased relative to the control.
On the other hand, Immunofluorescence is generally performed for localization and/or colocalization of protein in cells. As you mentioned, Immunofluorescence indeed looks beautiful. But you need to consider your end goal i.e., changes in the levels of an intracellular protein in the experimental condition. Western Blot is more sensitive and reliable than fluorescence intensity quantification.
Best.
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I am looking for an accepted (and commercially available) human endothelial cell line as an alternative to HUVECs to perform extensive cell culture experiments.
Many thanks
Karsten
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You may consider the following cell lines.
HMEC-1 is an immortalized human microvascular endothelial cell line.
ECV304 is a spontaneously transformed line derived from a Japanese human umbilical vein endothelial cells (HUVEC) culture.
EA.hy926 is a immortalized human vascular endothelial Cells.
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After Isolation of Kupffer cells from steady state mice, I distinguished KCs from Endothelial cells and macrophages were also ly6c positive, F480 positive, cd11b positive. The ly6c positivity is something I didnt expect at least in the steady state. Is it possible that macrophages are soautofluorescence that seem positive ?
Thanks a lot in advance
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Hello, I would like to ask what might be the reason why the Kupffer cells isolated from the mouse liver did not adhere when cultured with DMEM containing 10% serum, and how to improve it? Looking forward to your reply, thank you very much!
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I'm growing HUVEC cells in complete media called Endothelial Cell Growth Medium 2 (PromoCell) supplemented with Endothelial Cell Growth Medium 2 SupplementMix, as suggested by the company.
However I would like to know if anyone of you is growing HUVEC cells in other media that work good for HUVEC.
Thanks in advance for your help.
Federica
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I'd like to tell you about my experience with HUVEC (from ATCC).
As the basal medium, I use F12K nutrient mixture supplemented with 10% FBS, 0.1 mg/mL heparin sodium salt, and 0.03 mg/mL endothelial cell growth supplement (ECGS). This time, I'm using the entire medium by combining all of the elements listed above and changing them every two to three times per week.
This approach, I believe, will be beneficial to you.
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Hello!
I was wondering how many Human Endothelial Cells/well are usually used for MTS assay using Human Endothelial Cells!
Thank you for your time!
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Hello Woori Lee
You will have to optimize the seeding density by running a pilot experiment with different cell densities. If you are using a 96-well plate for MTS assay you could try various seeding cell densities starting from 2000 cells per well and go up to 20000 cells per well. The seeding density will depend on the cell type and its growth rate as well as the number of days of your experiment.
Choose a cell density that will be at least 70-80% confluent on the day of treatment, so that they do not get over confluent on the final day of the result as this could affect your readings.
Best.
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I'm doing a preliminary experiment to induce CXCL9 via TNFa in Endothelial cells. The plan for the experiment is to bring up some Endothelial cells and expose them to TNFa (as we already have this in stock else we would use IFN-y). Has anyone got any experience in what concentration of TNFa and the duration you would use to start this experiment or could point me to the literature that might be useful?
Many thanks for reading.
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Dear Calum,
20 ng/ml TNF-a typically result in a strong activation of the NF-kB pathway in endothelial cells and activation of target genes. Depending on the gene, you may see the strongest induction after 3-6 h but in some cases also later, e.g. after 24 h. However, in case of CXCL9, I doubt that endothelial cells will upregulate this gene very much. If at all, you will probably see a better response using IFN-g (100 ng/ml) or a combination of TNF-a and IFN-g.
Best, Lothar
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WE use ZO-1 - but see weak signal with hbec51 cells
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Another classic pan-endothelial marker that should distinguish these cells from glia is Ve-Cadherin.
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I was wondering if anybody has a good recipe they use for a buffer and enzymatic cocktail they use in order to isolate microvascular endothelial cells from small intestinal tissue from a rat.
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Hello Chris Delavan ! I was wondering if the protocol mentioned here for mice, worked for your rats? or did you have to do adjustment? My experiment led to low cell counts for rat intestine when I used the mice protocol, so was wondering if yo have got it to work?
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Hello, I would like to ask some question. We were testing on Nunc 3 micro pores inserts the transmigration of monocytes through endothelial monolayer, which was made by seeding endothelial cells (HAEC) on the membrane. To test the transmigration, we added soluble Endoglin to the bottom compartement in media after 100% confluence of the cells in the upper compartement. The soluble endoglin is cca 65kDa big protein. We would like to know, if it (soluble Endoglin) can travel through the monolayer from bottom to upper compartement to propose results. Thanks a lot for your help, we will appreciate it.
Kind regards, Mgr. Martina Vasinova, Charles university in Hradec Kralove
Product Web Id: 140663 Product Title: Nunc™ Polycarbonate Cell Culture Inserts in Multi-Well Plates Product Line: LSG_423
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Hi Gediminas,
thanks a lot for your detailed answer, you gave us some tips to test in our lab.
I appreciate it a lot !
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So I'm trying to use a program to count the amount of cells that are in the corneal endothelial cells like in the image below. I have tried using ImageJ with the threshold and analyze particle method. However, after sharpening the image and removing all the parts of the image that wasn't endothelial cells I still can't get the program to work well. As the boarder between the cells is either too little so it counts a lot of the cells as one, or the cells are fragmented into little pieces so it counts one cell as more than one cell.
Also the image below is diseased, so the black spots aren't part of the endothelial cells.
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These corrections may help you.
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Hello,
I would like to ask your opinions on an interesting case:
46 yr old female patient had an infection of uppper respiratory system in the beginning of September. Shortly afterwards she developed angina pectoris like symptoms. EKG was without pathological signs. In 24h-EKG only a sinus tachycardia showed, when walking slowly (up to 140-160 beats /minute). Echocardiography was without pathological signs as was lab (CRP, troponin, myoglobin, hemoglobin,...). In a cardio-MRI in November a cardiac microvasculatory dysfunction (endothelialitis? small vessel disease?) was diagnosed.
In a blood test, troponin, creatine kinase, creatine kinase-MB, CRP, c-ANCA, p-ANCA, lupus anticoagulant, anti-cardiolipin antibodies... and so on all were negative.
Only antibodies against endothelium could be shown.
Does anybody here have experience with a case like that? At the moment, we think mainly about an autoinflammatory process (e.g. triggered by auto-antibodies). Is there any way to find evidence pro or con?
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Thank you for your answers!
I don't have experience with a case like this, but it seems plausible that they could be related with this manifestation. I suggest the following literature:
Best regards
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I need help with primary mouse brain endothelial cell isolation and cultivation. My cells do not attach to the tissue culture plate (coated with fibronection) after overnight incubation. They are not stained by Trypan blue prior to seeding.
1. Instead of using adult brain dissocation kit (Cat# 130-107-677), I used neural dissocation kit (Cat# 130-092-628).
2. For all the centrifugation, I used 400g x 7 min, instead of 300g x 10 min.
3. I didn't perform RBC lysis. Instead, I performed transcardial perfusion before dissecting the brain.
I also have a question regarding CD45. The protocol suggest removal of CD45+ cells with negative selection. Is that truly necessary?
Thanks!
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You can try using the method described in the attachment provided.
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I'm trying to isolate endothelial cells from mouse brains for culture and I've tried a variety of protocols with mixed success and was hoping for some pointers. I've had no issue with the isolation/digestion of the capillary fraction but when it comes to separating the capilliaries from the debris/erythrocytes I've had less luck, this is what I've tried so far:
1. Horizontal pasteur pipette which allows the debris to 'settle' so the endothelial cells can be squirted off. This was the original lab protocol and I've found it very ineffective.
2. Percoll gradient. This was from a JOVE video and is a gradient set up at 3000g for an hour using 10x PBS, 1xPBS, FBS and percoll. The capillary fraction is layered on top and spun for 700g for 10 mins (no accel/brake) then the cells/capillary fragments are supposed to be in the interphase. This resulted in zero cells in the interphase and a lot of debris pelleted right at the bottom of the tube, very much not what was on the video.
3. Optiprep gradient. Pretty much same as above but I used optiprep because the percoll hadn't arrived and I had it in the lab. This time I ended up with a diffuse pink layer (medium), a clear interphase (supposedly cells), a pale red band (erythrocytes/debris?) and a clear layer. I tried the interphase - no cells, then I tried the red band which got me capillary fragments as required but also tons of what I assume are erythrocytes (very small cells...?)
Anyway, I'm wondering whether anyone has any recommendations for getting a cleaner vessel fragment fraction? The one time I have managed to get a very dirty fraction the fragments did grow into endothelial cells, I'm just wondering whether there's a cleaner/better way to do it by modifying my gradients somehow?
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Hi Gediminas,
Thank you so much for sharing your protocol. I still have some details to ask:
- What age are those mice?
- So you don't remove myelin or RBC, right?
- Roughly how many cells can you obtain out of 6 mouse brains?
- How long does it take for the extracted endothelial cells to attach after plating?
Thanks you!
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HI all, any suggestion on how extract proteins from HUVEC? Now I am getting about 0,5 ug/ul from a confluent 10cm dish. Currently I am using a buffer with 1%NP40, 250mM NaCl, 10% glycerol and 50mM Tris-HCl pH 7.5 + proteinase inhibitors.
Thanks!
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I have been trying to pull down Ras homolog family member A (RhoA) from HUVECs and running into the same issue. It was challenging to detect RhoA with 30ug lysis cells on the western blot. What is the highest concentration one could expect when extracting protein from HUVECs? Any hints are appreciated.
Dimitris Basagiannis
Alice Gentil Dit Maurin Fabrizio Renzi
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Hello!
I'm trying a new protocol, and will be seeding isolated mouse endothelial cells on a 0.1% gelatin-coated 12-well plate. I was wondering if there were any established methods to harvest the cells once they have reached confluency? I normally scrape the cells to harvest, but am concerned that this may not be a viable approach with a gelatin coating.
Thank you!
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You can use accutase, lidocaine, versene...
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Hi,
I want to quantify the cell area of bEnd.3 cells (mouse brain endothelial cells) using confocal microscopy by ImageJ. Most of the available protocols need to ignore neighbouring cells where cell-cell boundaries are in touch with each other. However, endothelial cells show continuous morphology. I will be really grateful if someone can help me with the protocol for cell area calculations using ImageJ.
I also want to quantify the distribution of tight junction proteins in cytoplasm vs. plasma membrane of these cells. If there is any way by which this quantification is possible using ImageJ then please help me.
Any help is appreciated.
Thank you in advance.
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If you visually cannot distinguish single&full cells, even with lower magnification, then I am afraid that you need additional staining that evenly stains the membrane to quantify cell surfaces (as I have mentioned above) so the boundaries between cells are clear. Also, nuclei staining would also help to distinguish single cells. PS: General rule: one can only quantify things that are visible.
Regarding the delineation of membrane and cytoplasm compartments - same rule. You can only measure/delineate if you see it, so I am afraid that lower magnification than what you showed above, would not allow that. Also, as already mentioned you have no marker for the membrane, so the delineation will not be very accurate. You would need additional staining of the membrane on top of your protein of interest to be accurate.
Furthermore, is there a way to seed the cells at lower density? This could greatly help your analysis as maybe then you can easier distinguish single cells.
Good luck!
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I am currently working with primary human retinal microvascular endothelial cells (HRMECs) and need to grow tight confluent monolayers for TEER, transwell permeability assays, and TJ/AJ immunostaining. At the moment, my cells appear to grow in a 'patchy' manner, with some areas of high density and others where few, if any, cells grow at all.
Does anyone know of any ways which might result in growth of uniform, tight, confluent monolayers? If I leave the HRMECs in culture too long then they just start to die, rather than reach confluency.
I know some of these primary endothelial cell types have issues with contact inhibition and such. Is there any way to get around this?
Any advice would be greatly appreciated!
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Hello, when we culture primary porcine or rodent blood brain barrier cells, we
use from day 3 on culture medium containing 550 nM hydrocortisone, which results in higher TEEr values and morphologically better monolayers.
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I order protein VEGF121 (pepertech). but there CoA only mention about reconstitution. I wonder it can dissolve in media. In the manual, they just mention like "After initial reconstitution, further dilute in a buffer containing a carrier protein or stabilizer (e.g. 0.1% BSA). Store working aliquots at -20˚C to -80˚C "
Q1. Are there any method to dissolve in media? (basal media or full media)
Q2. Can store long term in media?
I want to dissolve in endothelial cell media (protech).
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Abha Sahni Thank you for your advice.
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I have used both viral supernatant as well as viral particles concentrated by ultracentrifugation to infect EA.hy926 cells. But I have been unable to transduce them. The same viral particles have been able to efficiently transduce other cell lines. I have tried both 8ug/ml and 10ug/ml polybrene, but have not been able to transduce the cells. Can someone please help me out?
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What is your measure of transduction success? Transgene expression, surviving rate of stable cells during antibiotic selection? May be it is not the transduction itself, but something else.
Have you tried different MOIs and repeated transductions? Some cells need higher virus concentration.
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Dear All,
I was setting up the first time a scratch assay using endothelial cell line HMEC-1 and observed HMEC-1 cell shrinkage/detachment after 9 hrs into the assay.
Endothelial cell line were confluent at the time of assay, grown on 24 well plate in Endothelial Growth Medium MV from promocell. The wound on endothelial monolayer was created with a 200uL pipette tip (ibidi inserts should allow better consistancy), followed by 2 rounds of PBS wash (for cell removal). Fresh endothelial growth medium was then added back to HMEC culture before live cell imaging at 37C, 5% CO2. I checked the CO2 and temperature reading throughout the assay and they were both stable during the course of the assay.
Could anyone kindly suggest what might have gone wrong during the set-up and what might be the cause of that.
Many thanks!
Tommy
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Does it work not washing the cells or changing the medium?
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Which connexins do bEND5 cells express? Are there any special culture conditions required for them to form gap junctions?
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Please check the attached paper.
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I am about to work on a research project using endothelial cells and I have some doubts:
- If you have to choose two or three among the available characterization markers, which markers would you pick?
- Is flowcytometry superior to other methods in terms of cell characterization?
Thanks in advance, any experiences shared here would be much appreciated
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It also depends on the type of endothelial cells you plan to characterize. For instance, for cerebral microvascular endothelial cells of the BBB you want to use claudin 5, occludin, ZO-1 in addition to CD31.
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I try to transfect endothelial cells with firelfy luciferase plasmids under control of various promoters with Lipofectamine LTX, but transfection does not work at all, does any one have any idea how to transfect these endothelial cell lines ?
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ok, thank you, we will send you soon a request
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Hi, guys, I want to study two genes' function in endothelial cells at the same time. I would like to use siRNA to knock down one gene and lentivirus to overexpress another gene. Do you have any suggestions or established protocols that I can follow? Another issue is that I want to use these cells for glycolysis seahorse assay later, should I do the siRNA first then overexpression or overexpression prior to siRNA knockdown, because the microplate will be used. Thanks in advance!
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In principle that should work fine, I’ve done similar before. Obviously make sure to run appropriate controls to monitor possible effects of each treatment on the other. This ultimately depends on which gene you are knocking down and which you are over expressing.
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Hello,
I am interested in sorting out tumor associated endothelial cells from an orthotopic lung tumor model for the purpose of ATAC seq. I need atleast 50,000 cells and 90% viability for ATAC.
Does anyone have experience in this?
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Hi Anusha Acharya we are offering ATAC-seq end-to end services at Diagenode.
Let me know if you want to try.
Jessica
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It does not adhere to the plate and floats around. We are culturing endothelial cells and fibroblasts.
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Hi Nivedina,
This is rather interesting/unusual phenomenon (if it is related to a very few cells in the dish)! As mentioned by the other experts, the uptake of various substances present in the serum by cultured cells may cause them to appear dark (using the phase contrast microscopy). However, if this would be the case, then more cells should appear dark, as well. From my experience, senescent/apoptotic/dying cells also appear dark under the phase contrast, however, looking at your cells it doesn't seem that this may be the case. Also, I am curious if you have used any dye to stain cells during previous propagation etc.
It would be interesting to know if the "dark" cells proliferate and maintain this phenotype (assuming your cell culture is bacteria/fungi-free).
Best of luck!
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Hi all,
I am planning to use 1-2% methyl cellulose (15 cP, Sigma) to increase the viscosity of my cell culture media (EGM, Sigma). In the website of the sigma, they describe 2 methods to prepare methyl cellulose solution in water:
In both methods, water needs to be heated over 80 oC to dissolve methyl cellulose in water. Since I will be using cell culture media to dissolve methyl cellulose, I wonder if there is a 'milder way' to prepare this solution?
Thanks in advance for your suggestions!
Utku.
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Hi Utku,
Did you ever figure out how to prepare the media?
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Does anyone have success with any coating material (fibronectin, gelatine etc.) for HBMVEC and also be able to measure TEER reflecting a monolayer? I've been using a mix of matrix proteins and TEER is very low.
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what media is recommended to growing HBMVEC cells?
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I was wondering how long you can store samples and organs or tissues of mice under freezing conditions. I would like to use mice in my experiment and I will harvest the aorta, specifically aortic endothelial cells. I was wondering whether it is possible to store it for 1 year..
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If you plan on isolating the cells, you cannot freeze the tissue. The ice crystals will destroy the cell membranes.
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Hi guys
I'm attempting to isolate primary endothelial cells, and am having trouble with pericyte contamination of cultures. The endothelials look nice and have CD31 etc, and I've been identifying pericytes with PDGFR-beta. However, according to the literature, endothelia can be selected from a mixed population using puromycin which they can pump out, but is toxic to other cell types.
I just tried a concentration-response with my cells (10 ug/mL - 10 ng/mL), but at the higher concentrations where it is reported to be toxic to non-endothelial cells I saw the reverse (ie. pericytes survived and endothelia died).
Has anyone else tried puromycin selection of endothelia in human cultures and if so, what works/what is going wrong?
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Thank you Leon Smyth for your kind reply! I will find another alternative to purify my preparations.
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Dear all,
For the validation of a CD34 polyclonal antibody using flow cytometry I'm using BUVEC (bovine umbilical cord vein endothelial cells) as a positive control.
After validation I only have around 5% CD34+ cells from BUVEC, so I'm not sure it is because of the low percentage of CD34+ cells in BUVEC or that it is because the antibody for CD34 is not working.
Does anybody has any experience with CD34+ cells from BUVEC? Or other bovine sources f.e. aorta?
Thank you for considering my question!
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Hi Emma,
I would check the reactivity of my antibody.
Thanks
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So I want to isolate endothelial cells with a through positive selection. And after that do another positive selection to isolate the lymphatic endothelial cells, so the vascular and lymphatic endothelial cells are seperated. Is it possible to do this with MagniSort, and thus remove the magnetic beads after the first isolation?
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Hey Tess. Which marker do you use to isolate your endothelial cells? In case you use (human) CD31, I can recommend the Fab-TACS® system for the pre-isolation of endothelial cells. Instead of high affinity antibodies, low affinity Fab fragments are used. They completely dissociate from the cell surface after isolation. You also don't need a magent for this first step, so a second positive isolation round using magentic beads can be done and you don't have to worry about remaining magnetic beads after the first selection round. If you're interested, you can have a look here: https://www.iba-lifesciences.com/cd31-fab-tacs-agarose-column-starter-kit-human/6-3216-002
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Hello! I want to isolate RBEC from a rat, I used the protocol - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208856/. However, I used 5-day old rats instead of adult rats. Subsequently, the addition of puromycin (4 μg / ml) killed 100% of the cells. Could this be due to differences in the age of the rats, or is it worth looking for other errors?
Attached is a photo of a mixed culture, no puromycin.
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Dear Georg!
Please You look at the article and protocol:
Culture of rat brain capillary endothelial cells This method has been adapted from previously described techniques (Roux et al. 1989; Szabo´ et al. 1997; Deli et al. 2000). Briefly, 2-week-old Wistar rat cortices were dissected free of meninges and minced. All animals were treated according to protocols evaluated and approved by the ethical committee of INSERM, France. The dissection was performed on ice and in phosphate-buffered saline (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4,8mM; KH2PO4, 1.5 mM). The cortices were cut into very small pieces (1 mm3 ), homogenized with a 5-mL pipette and digested in a mixture of collagenase/dispase (270 U collagenase/mL, 0.1% dispase) and DNAse (10 U/mL) in DMEM for 1.5 h at 37C. The cell pellet was separated by centrifugation in 20% bovine serum albumin/DMEM (1000 g, 15 min) and incubated in the collagenase/dispase mixture for 1 h at 37C. The capillary fragments were retained on a 10-lm nylon filter, removed from the filter with endothelial cell basal medium supplemented with 20% bovine plasma-derived serum and antibiotics (penicillin, 100 U/mL; streptomycin, 100 lg/mL) and seeded on 60-mm Petri dishes coated with collagen type IV (5 lg/cm2 ). Either 4 lg/mL puromycin was added for 2 days or 3 lg/mL puromycin for 3 days. Puromycin was then removed from the culture medium and replaced by fibroblast growth factor (2 ng/mL) and hydrocortisone (500 ng/mL).
Of course, the more cells are obtained from a younger animal, the more sensitive they are to antibiotics.
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I am not looking for glomerular endothelial cells.  Is there any alternative endothelial cell that I can use instead of renal peritubular capillary endothelial cells? Thank you.
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Hi, did you get the peritubular capillary endothelial cells in the end? if not, could you please tell me what kind of alternative cells you used
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Published work has shown that Vav-iCre can mediated gene deletion in the endothelial cells, but in our own experiment, we found no significant deletion efficiency in the KO intestinal endothelial cells. Is that possible that Vav-iCre mediate different gene deletion efficiency between fetal and adult endothelial cells?
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Hi Tao, to best of my understanding, vav-cre is more specific in hematopoietic cells. You may try villin cre.
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Hi everyone,
I have observed the H&E lung sections of SARS-CoV-2-infected cynomolgus macaques. I realized that the endothelial cells were much activated in lungs and I want to score the level of endothelial cell activation in these H&E lung sections. I also want to compare with H&E lung sections from influenza virus-infected macaques. Do you know any scoring system for estimating the activation of endothelial cells in H&E lung sections ?
I appreciate your answer.
Sincerely,
Cong
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I can really recommend you to start a collaboration with a pathologist at your clinic. We faced the same issue last year with HE stained brain tissue. The consulted neuropathologist could score tissue changes with amazing speed and high details. Don't hesitate to directly write experts, worst thing that can happen is that they don't reply and from my experience people are happy to help.
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Hi everyone,
I am a first year PhD student working on amyloidosis. I would like some tips and tricks to crack efficiently my current nightmare, i.e. the western blotting of the Amyloid beta and its oligomers. I am using brain endothelial cells to understand its presence in different oxidation condition, but as you might understand I have some problem with the WB technique (I am very naive about it).
I am using 16% tris-tricine gels, but my 2X Bio-Rad loading buffer contains Coomassie so, when i transferred my proteins to a 0.22 um NC membrane it stained horribly all the membrane, making impossible the detection of the amyloid-beta antibody with any detection method. Then another problem I got is that, since I need to load huge amount of proteins (around 60 ug), I had to overload my wells and the electrophoresis was horrible. All my bands were so thick and not sharp as they should be (or I am used to with tris-glycine gels).
So, do you have any advice on how to run effectively this kind of protocol? Also, any advice regarding how to obtain more concentrated lysate would be beneficial. At the moment my cell lysate has a concentration of around 2.80 ug/ul.
Ps: I am using a cerebral cortex human brain lysate as a positive control, together with synthetic AB40.
Thanks!
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Your protein concentration is not bad; I think it should work. Please follow the protocol exactly as described in the attached file. You may use the URL therein to watch the method video.
All the best!
Anoop
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since CD105+ Endothelial Cells are CD31 negative, how do we differentiate them in cell culture from mesenchymal cells? is it using vwf?
thanks a lot
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Endotelial cells are negative for CD73 (and mesenchymal cell are positive).
There is a more complete discussion in this other question:
However take into account that in Mesenchynal cells culture conditions, even if there were some endothelial cells, they will eventually be lost through passages.
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I detect the protein expression of enos(nos3) in huvec and cannot get results with enos.The expression of  other proteins are strong enough to detect .The only one cannot be detect is enos . 
I extracted the total protein of HUVEC. Also, the final concentration of total protein is about 5mg/ml. 
So ,I need anyone who detected enos in huvec by western blot to help me .
Thank you for your attention .
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Andy Wijaya Hi Andy, I´ve got fine results with HUVECs by using 10 µg protein/pocket, 10% SDS-Gel. I used eNOS antibody from CellSignaling (D8A6N) #35362 at 1:500 in 5% BSA, incubated overnight @ 4 °C. Secondary antibody was diluted 1:5000.
Good luck!
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Hello
I am new to work on endothelial cells, and I have been reading about them a lot and trying to know the difference between human umbilical vein endothelial cells (HUVECs) and bone marrow endothelial cells, and was trying to know why HUVECs used most in research comparing to bone marrow endothelial cells.
I couldn't find much information about bone marrow endothelial cells especially to know the difference, so would any who has any experience in this kindly help me? Would be a great help.
Many thanks in advance!
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Our lab harvests HUVEC (human umbilical primary endothelial cells) and endothelial cells can support COVID19 growth. The mothers will be screened for Covid, but we all know that there are false negatives, particularly in very early or very late stages of infection. Does anyone know of a kit that could be used to detect contaminated cell lines?
Thanks,
Naiche
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May be
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I'm looking for a culture protocol for MS1 cells (Mile Sven 1, ATCC: CRL-2279 ). This is an adherent endothelial cell line isolated from mouse pancreas. ATCC has a protocol, but it's lacking details on:
1) How often should they be passaged
2) How many cells should be seeded in a T75 flask
Thank you in advance for any help
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Pia Nyeng you should maintain cells at between 30-80% confluency. Any less will induce a lag phase as the cells adjust to their surroundings. Any higher will induce a stationary phase as cells run out of resources and space.
My recommendation would be to allow your cells to grow to 80% confluency, and passage at a 1:8 ratio. This gives you three cell cycles for growth (2, 4, 8). If you know how long your cells take to divide, you can multiple this by three and come up with a protocol as to when to passage your cells.
I hope this helps!
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I am working on the capillary formation of Lymphatic endothelial cells in a collagen matrix. But I am facing a technical problem to handle Collagen. I have made many times; I am not able to remove the bubbles from the collagen. After 2 hours of gel solidification of collagen gel, It is showing dark color in place transparent matrix. 
If anyone gives suggestions to resolve this technical problem, I will be grateful.
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Dear Devendra!
Please You look at this article:
Protocol
1. Neutralization, Dilution and Polymerization of Collagen Solutions for 3D Investigation and Cellular Contraction Assays
3. Generation of PDMS Microchannels for Collagen Fiber Alignment
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I am working up 60 million RAECs for cell injections. I was wondering how to best time my passages in order to produce the desired number of cells without having to freeze them down prior to injection. 
Does anyone have experience culturing these cells or know where I might find a baseline mitotic rate from which to plan? 
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Hello,
I would like to purchase an immortalized cell line for pulmonary microvascular endothelial cells (MVECs), but I could not find any. I looked up websites, like ATCC, Lonza, Cedarlane, and etc., but all I found is primary cells. Can anyone help and let me know if they have bought a cell line for MVECs, please?
Thank you so much!
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ATCC is distributing the SV-40 immortalized HuLEC-5a. See:
You probalby can get HPMEC-ST1.6R (SV-40+hTERT) from James Kirkpatrick group at Mainz University:
Good luck
Amos Bairoch
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I am culturing 'Human Umbilical Vein Endothelial Cells' & 'Normal Human Lung Fibroblasts' from the PELOBiotech in their 'Endothelial Cell Growth Medium' & 'Fibroblast Growth Medium'. I am wondering whether it would make a difference if I used the 'Accutase solution' instead of 'Trypsin' for cell detachment! Any experiences? (For the HUVECs, I am using Collagen-I coated plates)
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hi
Don't worry, it doesn't matter.
1-For FACS assay "accutase " better than " Trypsin-EDTA "
2- For general application " Trypsin-EDTA " stronger & cheaper than "accutase "
good luck
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I am focusing on the angiogenesis and trying to start tube formation test. I have tried some protocols on primary HUVEC, but met some troubles when I want to keep it by successively passaging. Can I use cell lines for tube formation? I want to try human umbilical vein endothelial cell (HUVEC) and human brain microvascular endothelial cell.
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Hi, as Yi Yan said it's no problem.
This Angiogenesis Assay Kit (i.e. an endothelial tube formation assay) works well with both types of endothelial cells - primary or cell line:
The troubles with the HUVEC were that they stopped proliferating after a while, I guess? Unfortunately a feature of primary cells:
This blog article " The Right Choice of Endothelial Cells for Your Vascular Research " might also be of interest:
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In an ophthalmology research involving endothelial examination by specular microscopy, there is a lack of correlation between the endothelial cell count and cell density, as is shown in the figure.
The results of specular microscopy examination on the 3rd month post-op shows that the mean endothelial cell count of the "red" group is lower than the "black" group, while at the same time the cell density of the "red" group is higher than the black group.
I expect that the inherent correlation between "count" and "density" would mean that one group would rank higher or lower regarding both variables; but I'm thinking that I might be wrong.
I am not an expert in specular microscopy, so I would appreciate it if someone could tell me if the data presented in this study can't be right and is impossible or is there a scientific explanation for this lack of correlation?
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Thank you James Leigh for your answer. In this case, if the area scanned is not the same in all patients of both groups, then the results of cell count are statistically meaningless. Thank you for your time and input.
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I am having a hard time isolating coronary endothelial cells from SHR rats. Previously, I isolated coronary endothelial cells from SD rats and used EGM MV media from promocell. For SHR rats, the cells are not attaching. I tried both EGM MV and EGM2 media from promocell and it didnt work. Looking for suggestions.
Thanks
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Alexander Trampe Yes, I am using gelatin pre-coated flasks. Thanks
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tldr;
1)Does anyone know of a difference in 2-NBDG and 6-NBDG in MS1 cells compared to other cell lines?
2) what is the best method for glucose uptake analysis in ImageJ? Is there a better/easier/not too expensive way to analyze glucose uptake in MS1 cells using 2-NBDG?
Hello,
I am working with MS1 cells (immortalized mouse endothelial cells) and I am trying to measure the glucose uptake using incubation with 2-NBDG, imaging in light fluorescent microscope and the analyzing the images in ImageJ to determine glucose uptake.
I have encountered several problems of which I wonder if anyone recognize or perhaps know how to fix.
1) First we used 6-NBDG which was appealing because of different reasons (presumably less quenching etc) but compared with other cell lines we needed much more 6-NBDG to get a signal in MS1 cells. We tried a couple of times and also with another cell line but then we switched back to 2-NBDG which had been used previously in the lab. 2-NBDG gives an okay signal at concentration of 100 mikrog/ml. We wash once with warm PBS, then incubate with 2-NBDG for 20 minutes 37 degrees Celsius and then wash three times with cold PBS before imaging.
Has anyone encountered this problem and knows any possible explanation?
2) the more important question regarding the analysis.
In order to approximate the glucose uptake we take images using a ligth fluorescent microscope. 4 images per well in 48 well plates. The pictures are then manually analyzed in ImageJ by thresholding and taking the histogram into excel where we have a template for calculating the percentage area of fluorescence (=glucose uptake).
Some problems I have
a) there is sometimes much bakground which disturbs ImageJ a lot, this would I assume become better if the cells were like 100% confluent. As of now they are approx 80-90% confluent. Confluency in itself affects the glucose uptake which is also something to have in mind, but if all the samples have the same confluency I assume it should be somewhat compareable.
b) is there a "more correct" way to analyze the glucose uptake in imageJ other than thresholding and using the histogram?
c) has anyone used flowcytometry or spectrophotometry for MS1 cells and 2-NBDG?
d) has anyone experience using Bio7 and R to make the process of image analysis more efficient? Any general tricks?
Thanks in advance
Malin
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Hi Liao, sorry for the late reply. Unfortunately I can't answer your question. Maybe @Azadeh_Nilchian recognize the issue?
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Hello all,
I isolate HUVECs for my research.
Kindly suggest me an CD31 marker product code (from Thermo scientfic or Sigma aldrich) for immunofluorescence microscopic characterization of HUVECs.
Thank you so much.
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Dear Jyotsana,
I think the following product could be very efficient for your analysis: CD31 monoclonal antibody (MA3100). You can access to the product by this link: https://www.thermofisher.com/antibody/product/CD31-Antibody-clone-HEC7-Monoclonal/MA3100
Best regards,
Dave
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I'm trying to make a experiment in which i use fibronectin as a surface coat, but the cells are from Rat (C6 glioma and endothelial cells) and the fibronectin we have here is from human. Can I mix both?
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It should work with human as well as with rat fibronectin.
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I have Endothelial Cell Growth Medium, cat no. 211-500, Sigma-Aldrich. For the culture of Human aortic endothelial cells do I have to add FBS, antibiotics and other supplements with this medium which is written "all in one ready to use" in sigma website?
Please suggest.
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As per the manufacturers guidelines, you need not to add anything in the medium. the Media is supplemented with all necessary things.
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Hi everyone!
I'm currently using C3H 10T1/2 cells to act as pericytes/support cells for vessels formed in 3D by HUVECs. However, I see that people also use other cells (smooth muscle cells [SMCs], mesenchymal stem cells [MSCs], human normal dermal fibroblasts [HNDFs] etc). Has anyone tried and compared the performance of difference support cells?
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Hi John,
You can find some papers that have studied the co-culture of endothelial cells with supporting cells. Here, I attach that paper:
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Looking to use phospho-antibody (Ser177) for p-eNOS staining by flow cytometry in human umbilical vein endothelial cells.
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I am struggling to do pAKT expression on PBMCs for sometime. As you mentioned above, may i ask you for the protocol of the same?
Anticipating your response.
Thanks
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I'm trying to coculture HMVEC (human lung microvascular endothelial cells) and NHBE (normal human bronchial epithelial cells) on 0.4 micron transwell with PET membrane. I'm seeding endothelial cells on the bottom side of the transwell and epithelial cells on the top. However, my endothelial cells keep balling up on the bottom side. I'm not sure why this keeps happening. Is it the material that is not working for the endothelial cells?
In general, why do endothelial cells do this?
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Caroline A Owen Don Kaiser Hi Both, thank you for your really great answers! I’ve actually found out the answer to my problem and am posting in case you guys are also working on coculture. It was actually a simple problem.
We’ve been using Corning’s 0.4 micron pore, PET membrane transwell insert and as my post mentions, we’ve been seeding endothelial cells on the bottom of the transwell and epithelial cells on the top. The representative actually told us that the bottom side is not TC treated which was why the cells never stuck. Caroline A Owen actually, though what I noticed Was that the cells did grow out Even though the transwell was not TC treated. Don Kaiser we did coat the bottom side with 0.1% gelatin and the cells eventually did grow out. However, I don’t think that they will stay on for longer since gravity is not our friend and they will eventually get pulled off...
thank you for your answers! I appreciate it
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Hi everyone:
I tried to isolate the MLEC with collagenase type 2 and positive selection with LS column and CD31 antibody however, cells are more look like fibroblast rather than endothelial cells.
Does anyone have a protocol?
Thank you!
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Thank you so much
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YAMC (conditionally immortalized mouse colon epithelial cell line) can be used in
the endothelial cell tube formation assay in the study of in vitro study of angiogenesis?. Appreciate your inputs and any suggestions of research articles/protocols related to this.
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Dear fellow, Kedar Ghimire thank you so much for correcting me. He asked question for endothelial tube formation assay and while reading I ended up with angiogenesis. So I replied coz YAMC cell can use in angiogenesis assay when leptin use as inducing agent. I agree with you.
Thank you.
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Hi, I like to study high glucose affect in retinal endothelial cells, whether it is necessary to set experiment also with L-Glucose or Mannitol along with normal Glucose as Control.
1 2 3 4
Normal Glucose L-Glucose Mannitol High Glucose
Whether 1,2, and 3 will be my Control against High Glucose ?
Or just Normal glucose act as Control?
Like to have your valuable suggestion.
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I my opinion, a osmolarity control (Mannitol) is needed to allow a comparison of glucose-induced effects in this setting.
(For ref. see e.g. Langer et al., J Nephrol. 2016 Dec;29(6):765-773. doi: 10.1007/s40620-015-0258-1 )
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Does any body knows a protocol to detect internalization of a cell surface receptor like TLR4 or VECadherin in endothelial cells.
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Hello Shadab,
I used the anti-TLR4 primary antibody and secondary antibody IgG H&L (FITC labeled). Additionally, I used 4% formaldehyde (10-15 minutes) for fixation and 0.5% Triton-X100 for 15 minutes to permeabilize the cell. The cells were washed three times after every step with PBS-T followed by confocal microscopy. As of the time period, it will solely depend on your experimental design and cell-line. Therefore, you will have to standardize the period of experiment in time dependent-manner as
Mohammad Parvaiz Qamar Farshori
mentioned. For your information, I performed the experiment on H9C2 and C2C12 cell lines.
Best wishes,
Mahmudul Hasan
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We are successful in isolating endothelial cells from Rat lungs and heart. However, due to active endothelial-mesenchymal transition sub-culturing the isolated cells is challenging.
LONZA in their website guarantees sub-culturing human cardiac endothelial cells until P10. Is this true..? Does someone have any experience in culturing them?
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Can I please check if you were able to successfully passage these cells for 10 passages with no mesenchymal contamination?
Thank you!
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I want to compare how are actin fibers in two different context in endothelail cells and I am not very familar with actin. I would like to quantify both quantity and also distribution of the actin.
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You can quantify the intensity and positional information of F-actin inside the cell and the changes associated with it on using different actin disrupting treatment protocols.
I hope this paper will be of interest to you.
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The origin of many, if not all, vascular diseases can be correlated with endothelial cells (ECs) mechantransduction. The mechanosensory of ECs is strongly linked to the physics of blood flow. CFD models of vascular blood flow can provide insightful information about the hemodynamic environment associated with different vascular disease. Hence, CFD can contribute to the research on ECs mechanotransduction. However, since blood flow is inherently multi-harmonic pulsatile flow and vascular geometry have complex 3D morphology, CFD models require rigorous validation practice in order to ensure that their results are as valuable as they are considered to be.
I would like to discuss the previous efforts conducted to provide consistent, reproducible and physically meaningful validation procedure for CFD models of pulsatile flow for vascular hemodynamics applications. Is there any validation resource available for public access? What would a good and reliable validation database include?
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Thank you Dr. Kartik Jain for the informative response. I have used many of these validation datasets in my early CFD work on turbulent combustion modeling. However, pulsatile flow is quite different from fully developed flow, especially when it comes to transition to turbulence. The FDA nozzle benchmark is dealing with fully developed flow too.
As to the mesh convergence tests, of course it's a good way to establish benchmark for evaluating the numerical error in a CFD model. Similar to Richardson extrapolation or any of its variants, mesh convergence estimates the error resulting from discretization scheme. Validation, on the other hand, aims at estimating the error resulting from the physical model and assumptions in the governing equation (such as viscosity closure).
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Hello everyone,
I hope that someone can help me out with the following question.
What difference can be seen when using endothelial cells vs HUVEC cells in an angiogenesis assay?
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Basically, HUVEC are immature mixed cells. These cells grow fastly and are good for pure angiogenesis assays. However, if you want to perform translational studies, you need to extend your studies to human aortic endothelial cells (HAEC) or human coronary endothelial cells. Moreover, you need to select cells appropriate for study cancer in comparison to CVD. Ex vivo direct angiogenesis can be also studied by DIVA, see De Nigris F et al Cancer Research 2008; 68:1797-1808 and Casamassimi A et al. DOI 10.1007/978-94-017-9716-0_2 a chapter book of 2015 on angiogenesis. Best, Professor Napoli
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I want to compare primary HUVECs with my immortalized HUVECs. I have done microarrays and was wondering if I can somewhere (some database etc) find a list of cell type specific genes (which, in my case, would be endothelial cell specific genes). Any kind of help would be appreciated.
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Hi Muhammad,
CellMarker database (http://biocc.hrbmu.edu.cn/CellMarker/ or http://bio-bigdata.hrbmu.edu.cn/CellMarker/) is a database of cell markers based on tissue and cell type.
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Hello,
Why dead endothelial cells detach from glass or other surfaces, but dead neutrophils remain attached? Which surface molecules or which characteristics result in such behavior for these different types of cells?
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I'm not aware of any bio-molecular differences for your observation so I can only hypothesize that neutrophils, because of their smaller size, have more surface area relative to their volume and presumably their mass, so they might be less likely to detach. This might be especially true for glass substrates to which endothelial cells attach less-well relative to substrates with fibronectin or collagen. On the other hand, I think endothelial cells 'spread' more than neutrophils, so the argument could be made in reverse, suggesting there me a bio-molecular reason.
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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.