PC3 Aggregates in Matrigel(Not "hanging drop method") - (May/19/2007 )

Hi To all!

even if I know this forum since a couple of weeks, this is the first time I write

I have a problem building cell aggregates in Matrigel..Is someone of you familiar to this technique?

Briefly I use Matrigel 1:2 and 800 000/1 000 000 cells for each aggregate, making a spot of 2 μl in a m/w.

Then before giving 500μl of fresh rpmi +1%Glu,+Ab, wo FBS, I wait 20 minutes to let clot the matrigel.

After a couple of days some aggregates die, other give no response...



What can I do? are 20 minutes cloting time too much? but if I wait less most of my aggregates melt as snow in a hot summernight...

Could somebody help me?

Thanks a lot

J.M.

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Matrigel should be used as a very thin coat; we use only 20 µl of Matrigel per 4.2 mm plate (Matrigel solved as recommended by BD)

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Dear bearer
Thanks for answering me so quickly ;-)

I don't think I was clear enough in my explanation:

I don't use Matrigel as a layer between the cells, but I make a mixture of Matrigel and cells (2 μl matrigel for at least 800 000 cells). After I've put out the RPMI from the eppendorf I "knead" the pellet with 2 μl matrigel 1:2, then I make a spot on a plate in the m/w (we used to use Polylysine but the cells weren't too happy). I aim to have an aggregate where the cells degrading the matrigel, make a "corona".

Do you (or anyone else) have suggestions?

Thanks to everyone!

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If you let the cell suspension with matrigel aggregate then you will not have an even cell distribution.

Try to coat the plate with matrigel and dry the plate and then seed the cells. This might be better..

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Thanks scolix!

What about the RPMI(+1%Glu+Ab, no red phenol no fbs) I should add?Is 1/2 ml for the first 24h ok?Or else How, if you was me, should you clear up this task?

Thanks to all

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Hi Joop!

I don't know this techniq and not clear understand from your words what do you want to do. We used similar techique only to inject cells in polymerized Matrigel which was inoculated in mouse to investigate in vivo angiogenesis. If you want to analyse invasion as follows from your words "I aim to have an aggregate where the cells degrading the matrigel, make a "corona"." so why not to make well known experiment in cell culture inserts coated with matrigel? Why are you interested especialy in "aggregates" May you write more about this. I don't know what Matrigel ( source) you use and possibly you don't achieve enough Matrigel concentration to polymerized. If so you problem with cell die to my mind deals with killing you cells with excessive amount of connective tissue proteins and cytokines which turn cell regulation into apoptosis way. Also it sounds strange that PC3 feel not good on polylysine, may be some problems with your cells also?

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HI to all
This kind of technique should give a very clear visualization of the cell capacity to degrade the ECM.
I (try to) use 3 mw: one control, one without FBS and one with FBS but also with a specific drug.
In a couple of days I see a kind of corona (very similar to that of the Sun) but in the place of beams I see the cells migrating. In the well in which I give the drug, I (should, if I a made my homework well) see an inhibition: I see less cells in this corona then in the control corona.
Unfortunately of 12 wells the ultimate suitable aggregates are very low.
All the aggregates (+FBS;-FBS;+FBS,+drug) suffer in the same way, usually they die, or give no response anymore.

Crucial factors are in order of time, the number of cells/quantity of matrigel, The "kneading" and the polymerizing time of matrigel

Last but not least the polylysine: Although other cell lines are very responsive to Lys, these cells are stressed. The generation is 21 so I don't think there are problems with them..
Neither we got problems with PC3-Lu cells.

Thanks to everyone
My tutor was enthousiast about the matrigel coated plate method and asked me how to try this...
WOW!
C YA!

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We add matrigel (1:40 diluted in media DMEM-without any FBS or Gln or antibiotics) and then make sure its covered evenly and then remove it. Now let it dry for atleast 30-45 min.

Now you can seed the cells.

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Alternatively if you would like to separate process of plate coating with your invasion exp, you can dry coated matrigel 96 well or 24 well plate plate in CO2 incubator for 2 hour ( with open cover). Than keep this plate steril at 4C for week or longer when you need to make your exp. But before seeding cells you should rehydrate your coated wells with culture mwdia for 2 hours in CO2 incubator.

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