I'm trying to perform invasion assays in melanoma cell lines (i.e. Lu1205, Sk-mel2) by using matrigel-coated membranes and a Boyden Chamber but I had problems in getting a thin and uniform layer of matrigel.
Thanks in advance!
Personally, I would not recommend to try to coat Boyden Chambers on your own as my Prof told me that you have to ensure that the electrical resistance is the same in each and every Boyden Chamber and you have to prove this as otherwise publications might be rejected from reviewers. Therefore, it would be better to buy them ready from a company.
Migration assays (better off purchasing a commercial plate, expensive but it works), Thus, I agree with Daniela. Below is how we use them
Transwell migration assays were performed using a modified Boyden chamber. Briefly, 4.0x104 cells were serum-starved for 6h and plated into serum-free and GF-free medium onto 8.0 micrometer pore transwell PET membranes (BD Biosciences, Bedford, MA). Five hundred microliters of medium containing 2% serum and growth supplements were added to the bottom well. Non-migratory cells were scraped off 24h later and migratory cells were fixed in 100% methanol, washed, and DAPI stained. Experiments were performed in triplicate transwells and quantified by averaging the number of DAPI nuclei per 20x field of view counting 5 fields per chamber.
Dear Jerry, thanks for your protocol. Mine was looking pretty similar, but after a few tests I stopped it.
Personally, I have doubts of the whole assay and this is a reason why I stopped using it - if you add serum-starved cells in the top of your chamber and in the bottom well your medium with serum and growth supplements - within a range of probably 1 hour the medium of top and bottom is completely mixed. So the assay is at least not working as something like a chemoattractant assay.
Next point is gravity: cells will always follow the gravity and go from top to the bottom, even if they have to actively walk through the pores. And how do you distinguish how much invasion will really come from cells and how much comes from gravity?
I know that this type of assay is still very common and you find a lot of publications on it, but personally, I don't really believe too much on the assay. I prefer to go for microscopy with IBIDI chambers, 2D and 3D models are at least for migration already available and I hope that they will soon offer also something for invasion, where you observe the cells during the invasion process with microscopy and don't go for only some end point determination.
I agree with Jerry.
I also extensively use Matrigel Invasion chamber manufactured by BD Sciences (See the detailed protocol at https://us.vwr.com/stibo/hi_res/10369333.pdf). I found that commercial chambers will guarantee the reproducibility of the result, whereas your lab-made chambers could suffer from the inconsistency in the thickness of the coat. You could use a sample without any chemoattractant (e.g., serum) in the bottom well as a negative control.
Dear Ludmila Campos
I am attaching a detailed protocol from Methods in Molecular Medicine. Please read it carefully as it will help you definitely.
Best Regards
Ready-made coated chambers (e.g. BD BioCoat) are indeed standardized, yet you can't differ the Matrigel dilution. I have mostly coated the insert membranes manually by adding a drop of 20uL Matrigel dilution per membrane and then spreading it using a 100uL tip. When working with Matrigel, consider the following:
Always thaw the Matrigel on ice from several hours before exp
Upon receipt, after thawing, aliquot the entire received volume (10mL) into microtubes (I did 100uL/tube), also on ice. Thaw the number of aliquots necessary for one experiment and always thaw Matrigel once, do not freeze-thaw multiple times.
Use cooled tips and place the plates on ice. Dilute in ice cold serum-free medium.
Store Matrigel aliquots long-term at -20°C.
After coating, place the plates at 37°C for 1h.
Avoid air bubbles in the gel, don't press through when pipetting.
Test some dilutions on the cell line of interest, e.g. 1/3 to 1/8.
When in vitro applied, you can use the growth factor reduced formulation to minimize unwanted effects of cytokines and growth factors present within the standard Matrigel formulation. Specifically TGFß is known to be at moderate levels in Matrigel, which can interfere with your assay.
Regarding the effect of gravity: when working with mammalian cells, the micropores to be used are either 5um (macrophages) or 8uM (cancer/epithelial cells), much smaller than the regular size of these cells. Cells should never "fall through the pores". Unavoidable however is the meniscus formation in such assays, clustering cells together in the central part of the gel layer. A Cancer Res paper from a Beatson group described the inverse application, i.e. coating and seeding on the bottom side and making cells transit the membrane upwards.
It is rather subjected to variance because of the addition of a Matrigel layer, making it less user friendly than doing a migration assay, but after some practice, it'll work fine.
For quantitation, different methods exist. We have performed a comparison between these endpoint assays and a more recently developed impedance-based system for real-time monitoring of motility and invasion (PLoS ONE (2012) 7(10): e46536).
@ Ridhma: still, one question remains - how do you check that you really have the same thickness of the layer or basically the same electrical resistancy? In your protocol you write nothing about it - and this is really a point where you can struggle with reviewers..
Dear Daniela Bruennert,
As your concern, the matrigel is the key point in this experiment. Firstly, the experimental condition is on ice. Secondly, when adding the matrigel into the Millicell insert, I will add enough volume (100 ul) of matrigel to form the layer, this volume is definitely needed to form a smooth layer. Then, you should take out 70 ul matrigel from the insert, because 100 ul matrigel is too thick to cell invasion, and the 30 ul matrigel in the insert is enough to form the layer and to have the same thickness.
@ Daniela: if you refer to the thickness of the Matrigel layer on the RTCA plates and classic transwells, this was recalculated according to the surface area of the respective wells. The RTCA system is on a 96-well level, while classic transwells are on a 24-well level. We followed the manufacturer's instructions for coating the RTCA plates, which is 20µL/membrane. We applied the same Matrigel volume (20µL) per transwell membrane. Of course, this results in a layer with increased thickness in the RTCA than in the transwells. However, we recalculated the density of the Matrigel (dilution in SF medium), making the gel less dense in the RTCA setting than in the transwells. In conclusion, one can say that the thickness of the barrier was larger in the RTCA, but because of its lower density, cells showed comparable invasion kinetics with the transwell Matrigel assay, in which the barrier was thinner, but denser.
Hope this answers your question.
@ Daniella .. I suggest you can try with reverse migration assay by using a 48 well chemotaxis chamber, thus u can do a high throughput assay, avoiding the question of gravity.. since this would be against the gravity.
If you are using 24-well Transwells, 100uL Matrigel may be too much. You can apply a 1/5 to 1/8 or 1/10 dilution and add 20uL per membrane. Spread the Matrigel equally over the membrane area using a 100uL tip and gelify at 37°C during 1h, then it should be ok. Then you can also seed smaller amounts of cells (25000 - 100000).
Treat cells like you normally do in routine culturing (detaching, subculturing, counting, viability). I would suggest to use the normal growth medium as chemoattractant and indeed resuspend the cells in serum free medium. Resuspend cells thoroughly and check this under a scope to see if there still are a lot of clusters. The spreading of the Matrigel on the membrane using a tip always worked for me, but there is the issue of meniscus formation, which is always a trouble when manually adding Matrigel...so when assessing the invasive cells, don't go too much to the periphery of the membrane. What should be avoided is a sharp focal zone of invasive cells in the center of the membrane and no cells around this zone, this is typically due to meniscus formation where almost all Matrigel is gelified in the peripheral area of the insert.
Ludmila, I have used Matrigel invasion in an Essen Incucyte FLR microplate scratch assay, protocol is available on the Essen website. another department in your institution may well have one, many places see thepotential for charging other groups for use and make them part of a hirable facility now.
Katyayni, what concentration growth factor Matrigel are you using? I have used high concentration growth factor Matrigel with HT-29 colorectal cancer cell line in said Incucyte assay and found 2mg/ml to be too strong and the cells aggregated and died. 0.3mg/ml worked well for them. The Essen rep recommend not using less than 2mg/ml as you cannot guarantee enough matrigel is there for invasion to occur and I dont know how to validate whether its happening or not. Therefore Ive bought standard growth factor concentration Matrigel and shall now try that at a range of concentrations instead. The high concentration growth factor stuff is recommended for supporting cells you are implanting in vivo I now know, whereas std concentration should be sufficient for cell lines. You live you learn
Hi I have a question. In the Corning protocol they suggest to dilute the Matrigel in a coating buffer (Tris ph 8.0 0.01M and 0,7% NaCl ). what is the best thing to do dilute it in DMEM without FBS or in this coating buffer?
Bio-101 provides this protocol for cell invasion assay using Matrigel: https://bio-protocol.org/prep793
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