I am doing Transwell matrigel Assay on BT474 cell lines as follow
To coat trasnwell inserts (8um pore) I added 75 ul of 2mg/ml matrigel in serum free media and incubated in the incubator for about four hours. I took off extra matrigel containing media. 50000 cells that were starved overnight in 200 ul serum free media were seeded in the upper well and lower chamber had 400ul complete media. After 24 hour I do not see any difference under microscope. and my other question is
How much (amount conc) of Matrigel would be good for BT474 if anyone has used these cells for this particular Assay?
any other suggestion?
I am also trying to optimize wound healing Assay.For this Assay I used 5 billion cells (as per literature) in six well plate. Next day The wound was made by p200 (filtered tip). I am facing couple of problems
1-Around the edges cells are lifted and they make a flap and I can see some cells still attached underneath which are not 100% confluent
2-The wound is too wide to get images at 10X, and even by p10 I got a wound that was too wide to take picture at 10X and it does not get closed even after 144 hour (6 Days).
Any suggestion will be much appreciated.
Hi Abdul,
Invasion: what do you mean you didn't observe any difference in 24hrs? Do you mean no cells have invaded? In this case how did you assay for this? Staining the underside of the membrane or simply visually? Did you rehydrate the membrane with media before adding matrigel? The assay may require optimisation with some very invasive cells (e.g. MDA231), I'm not sure how invasive BT474s are.
Migration: In my opinion 7 billion cells is way too high a seeding density. Ideally you want to let the cells divide a few times until they reach confluence and then scratch. Seed 0.3-1e06 cells then let them reach confluence over a couple of days. If you scratch an over-confluent monolayer then you will get the "flap" of cells every time so I would definitely avoid over seeding.
If you have to use a tip to inflict the wound then be sure to scratch only once with a p10 (p200 is too wide). If you have access to an esson wound maker I would recommend using this. If not consider using an insert (link) to get a uniform scratch area. One of the most important things in a wound healing assay is to ensure wounds are uniform across replicates and that becomes very difficult when you are applying the wound by hand.
144 days is far too long to assay migration, in this time frame you will have to contend with cells dividing in the wound itself, not migrating from the edges. You should be looking at 12-36 hours maximum in low serum media if you want to assay cell migration and not cell division.
Hope some of this helps!
John
http://ibidi.com/xtproducts/en/ibidi-Labware/Open-Slides-Dishes:-Removable-Chambers/Culture-Insert-Family
Hi Abdul,
Invasion: what do you mean you didn't observe any difference in 24hrs? Do you mean no cells have invaded? In this case how did you assay for this? Staining the underside of the membrane or simply visually? Did you rehydrate the membrane with media before adding matrigel? The assay may require optimisation with some very invasive cells (e.g. MDA231), I'm not sure how invasive BT474s are.
Migration: In my opinion 7 billion cells is way too high a seeding density. Ideally you want to let the cells divide a few times until they reach confluence and then scratch. Seed 0.3-1e06 cells then let them reach confluence over a couple of days. If you scratch an over-confluent monolayer then you will get the "flap" of cells every time so I would definitely avoid over seeding.
If you have to use a tip to inflict the wound then be sure to scratch only once with a p10 (p200 is too wide). If you have access to an esson wound maker I would recommend using this. If not consider using an insert (link) to get a uniform scratch area. One of the most important things in a wound healing assay is to ensure wounds are uniform across replicates and that becomes very difficult when you are applying the wound by hand.
144 days is far too long to assay migration, in this time frame you will have to contend with cells dividing in the wound itself, not migrating from the edges. You should be looking at 12-36 hours maximum in low serum media if you want to assay cell migration and not cell division.
Hope some of this helps!
John
http://ibidi.com/xtproducts/en/ibidi-Labware/Open-Slides-Dishes:-Removable-Chambers/Culture-Insert-Family
5 billion cells sound like too many. there are generally only millions of cells in a t-75 flask. I would seed cells so that they are confluent in 3-4 days, this allows the cells to condition the media and for tight junctions to form. I would use the migration assay from www.ibidi.de. it has much higher reproducibility. I would need more information to help with migration assay, like what are you challenging with, how are you visualizing cells, and details like that.
www.ibidi.de
I guess the membrane is too thick for cell invasion. You can prefer to use 20-30 ul matrigel for each insert (i assume that you are using 24 well plate). For wound healing assay, be sure that the cultures are confluent (70-90%) 'as a monolayer' at the start of the experiment. You can use 100 ul pipette tips . Try to make a straight scratch gently and slowly while keeping the pipette tip under an angle of 30 degrees. To keep the wound widht limited try not to scratch so roughly , it can enlarge the wound or do not scratch so softly, you can left attached cells at wounded area.
And are you sure that you were using 10x? i recommend you to check the objective you used, because it is really hard not to get an image of a wound which was made using 10ul p.tips..
Hi Abdul,
Please try to apply less cancer cells, 100.000 cell will be good for 24-well plate (transwell).
Good luck!
Thanks for the suggestions!
I have overcome the problem of migration and invasion, but still need to figure out wound healing Assay
Sevcan, you are right I may need to use less number of cells and let them grow till confluency.
Regarding 10X yes I could not see wound even at 10X, not sure the reason, probably cells are not properly attached to the wells and when I make wound detaching cells are pulling off some additional cells.
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