Before seeding the cells into the 96 well plates do you grow them in a normal cell culture flask and do you harvest them with trypsin or with accutase in the end? One other factor to consider is the cell density in your final 96 well plates. Are you aiming for around 10000 cells per 100µL ?

Ok. In my experience there are few things that play role in a successful matrigel assay:

1. Batch of HUVECs, i.e. some donors just do not form tubes unless you have used a pooled cell population from several donors. Also, passage number is very crucial. If cell batch is the problem then you just have to try a different one.

2. Trypsinisation process is very important - once cells round up immediately dectivate trypsin.

3. Good diserpersion of cell pellet before cell seeding onto the matrigel layer. You need a single cell suspension before seeding.

4. Do you use a full growth matrigel or a growth factor reduced matrigel? Generally, HUVEC do not form nice tubules on a growth factor reduced Matrigel. Ofcourse the choice of matrigel depends on the objective of your experiment hence this is a parameter you might not be able to avoid.

5. Once you have seeded cells do not move plate about much, try to leave it in peace as much as you can.

I hope this helps!

We used the commercial tube formation assay (96 well format) by Becton Dickinson and followed the instructions - which worked fine for us.

I guess you are pipetting cells and then adding media with growth factors.............if so, this will make cells stay in on place on matrigel...........

Second mix the required amount of cells with your growth factors and media in an eppendorf tube and then add to matrigel.............

third the matrigel may be the problem................if it is not solid this can be a problem..............just take of the cap on 96 well plate and place the plate in normal cell culture incubator for 1hour that should help the gel solidfy the way it should...........

Good Luck and hope ur exps work ,.............chearsssssss

Keep the above suggestions in mind and to add something about ur particular problem,clustering instead of tube formations, may be occurred due to: first the number of cells and ur transfection reagents, if u used of course. the toxicity makes them to be clumped each other especially if the cells not confluent before u transfect them. second after trypsinization u have to mix very well with the neutrilization medium to avoid clustering. and then u have also think about the healthy of the cells in addition to their passage(early passage is always preferable for this particular tech).

Dear Zelia, in order to have the formation of new endothelial tubes you have to cultivate HUVEC with fibrobalst, in a ratio for example of 1:1, and grow them for couple of weeks in media that is half for fibroblasts and half for HUVEC. Change the media every other day. Hope it will help.

I used in the past the commercial kit of angiogenesis assay by Bekton Dickinson following instructions and I had not problems so I think the main problem is the Matrigel used.

Recently with a new Matrigel mixture (Sigma) I did not have the same performance in my experiments . I think for a problem of cell density. 15.000 per well preparing plate in this way: (50 microliter of Matrigel in a 96 well plate + 100 microliter of medium plus FBS and growth factors).

Good luck

I have some experience with HMEC-1 only. In regard with HMEC-1, from my recollection, the starting cell density is a little bit higher than yours and the culturing of HMEC-1 requires EGF and hydrocortisone. Hope these help.

We use 15000 cells/well of a 96 well plate. Cell count should be optimum and cells should be in single cell suspension. Try using accutase instead of trypsin, as it is gentle on cells.

We have no experience with EC but I can recommend Dr. Veronica Morandi from the University of the State of Rio de Janeiro ([email protected]). Good luck!

How many HMEC cells did you put in each well Zhihong????

Dear Zelia

I have perform HVEC tube formation assay using matrigel. I hope following points may help you:

-Be sure that your cells are in good condition, check the incubator to be completely normal, Use 10% FBS after thawing cells and if U conduct a serum free test gradually decrease serum percent,

-I had not use any growth factors so I cant help U in this case,

-Be sure that Trypsin has been washed out completely,

-Cell number is very important, we used 60000-800000 cells per well in 24 well plates completely coated with 90-100 micro liter Matrigel,

-DO NOT use cells just after defreezing, let cells to fit the condition at least a week,

In best condition U can see Tube formation even in Ur Flasks

Good luck!

as stated above finding the optimal cell density is very important and too less or to much cells could lead inefficient tube formation or clustering . Since you are in a 96-well plate I would setup a several cell densities to determine the optimal one. 5000, 10000, 15000, 20000 etc. cells/well. I'm adding an attachment with background on the tube formation chapter which is a extraction of book on angiogenesis assays, hope it helps.

I would recommend to use ibidi microslides for tube formation assay, as they have optimized surface to avoid meniscus formation and cell clustering in the middle of the well. 12000-1500 of cell per well should be optimal. From my experience with vasa vasorum endothelial cells, I would suggest growth factor reduced Matrigel and growth arrested cell (24-48 hrs with low or even without serum), but wash the cell after trypsin well. You can stimulate tube formation assay with any growth factor of interest and use the medium with 10% serum as a positive control.

Good luck! Also, remember that HUVEC are different from donor to door. If possible try another stock. The cord after healthy pregnancy and delivery is the best.

Evgenia Gerasimovskaya ([email protected])

I have experienced the angiogenesis phenomenon in cell culture for HUVEC and endothelial cells of rat's aortic rings. May be it's necessary to change the kind of gel.

which medium are you using? I used to add several growth factors to the medium, including VEGF. Alos, check that the matrigel iss the standard one, not the growth-factor reduced.

You may add a growth factor with angiogenic potency; we successfully used FGF-1 - and got tube formation very effectively.

this is how i do it

- plate 100 microliter martigel with out bubbles per well of flat bottom 96 well plate. allow it to sit for 2 hours

- make HUVEC or other endothelial cell suspension at densities 150,000 cell/ml, 300,000 cells/ml and 600,000 cells/ml EGM2. You should be relatively careful when u harvest the cells so that the cell death is minimal.

- plate 100-150Microliters of the cell suspension on to the already soldified matrigel carefully without bubbles in triplicate.

- wait for 12- 24 hours. u should see well formed tubes in most of the conditions.

good luck

Had anyone esperience with trasformed mouse endothelial cells? How manu cells per well in this case? What medium and growth factors?

I combined the DMEM and Hams F12 cultures together with ratio of 50% / 50% and then added: 1) 20% FCS (20% Fetal Calf Serum) including VEGF, 2) PBS, 3) gentamycin to them. For preparation the gel, thrombin and fibrinogen were combined together.

Thanks for all. I am going to try some other growth factors and different cell densities, I am using 10000 cells per well, but I have the feeling that cells get too dense.

thanks for your suggestions

Zélia

contact me via e-mail, I send you our protocols.

Kurt

I'm ready for presenting my experience to you too.

The problem can be caused by different factors as mentioned in previous posts. But I recommend - as Evgenia V Gerasimovskaya - to try µ-Slide Angiogenesis from Ibidi, because these slides are directly built fore tube test.

I use 20000/30000 cells/well in the 96 well plates.