I have tried to do transwell cell invasion assay and encountered a problem, in which after 24 hours of incubation in DMEM complete media (10% FBS) only a few cells (PANC-1 pancreatic cancer cell lines) was invaded through the Matrigel-coated cell culture insert (BD Biosciences, 8 um pore size insert, Cat no.: 353097), versus no cells invaded in serum-free media (SFM).
Following is the details of my protocol:
1) Thaw Matrigel (Corning, Cat no.: 356234) 24 hours in ice in 4oC refrigerator.
2) Dilute Matrigel to concentration of 0.4 mg/ml (from 9.7 mg/ml based on the Lot information of matrigel purchased).
3) Load 100 ul of Matrigel (0.4 mg/ml) into the upper chamber (cell culture insert) and incubate 24 hours in 5oC CO2 incubator.
4) After 24 hours, add 750 ul SFM or DMEM complete media (10% FBS) into lower chamber.
5) For upper chamber, add 200 ul cell suspension (2.5 x 105 cells/ml in SFM) onto Matrigel-coated cell culture insert and incubate for 24 hours,
6) After 24 hours, remove the medium in upper chamber and wash twice by PBS.
7) Fix cells by formaldehyde (3.7% in PBS) for 2 min at room temperature.
8) Remove formaldehyde and wash twice by PBS.
9) Permeabilize cells by 100% methanol (not ice-cold) for 20 min at room temperature.
10) Remove methanol and wash twice by PBS.
11) Stain both non-invaded and invaded with 0.4% crystal violet and cover the 24-well plate with aluminium foil and then incubate at room temperature for 15 min.
12) Remove crystal violet stain and wash twice by PBS.
13) Scrape off non-invaded cells with cotton swabs.
14) Count invasive cells in four random microscopic fields under a light microscope (bright field).
I wonder how come there are only less than 10 invaded cells in purple (that I can view under the light microscope). Following is my assumptions:
1) Maybe culture PANC-1 cells in SFM in the upper chamber made the cells static or less migratory. Maybe, I should prepare cell suspension in DMEM media containing 1% FBS.
2) Or, 24 hours incubation is too long that is why invaded cells has already been in the media of lower chamber instead of stick to bottom of cell culture insert's membrane.
3) Or, perhaps the Matrigel concentration (0.4 mg/ml ) is still considered high. Should try to use lower concentrations.
4) Or, before adding the cell suspension into upper chamber (after 24 hours of Matrigel coating) should rehydrate the Matrigel for 1 hour in 5% CO2 incubator.
5) Or,wash too harsh with PBS (but I just dip the cell culture insert inside beaker containing PBS) until detach the fixed cells.
Any idea to solve my problem in order to have more cells invaded through the Matrigel-coated cell culture insert?
Do hope to hear from you all in soonest time.
Thank you.
I think that investigating the time-dependent aspect of the experiment would be useful. Have you tried the experiment by stopping and quantifying the cells after two, six, twelve and twenty-four hours incubation?
If the problem is with other conditions, there would be an easy way to test. Keep the matrigel and media conditions identical to your previous conditions, but find a way to add a chemo attractant to the transwell insert that works for your cell line to induce migration. I've never used matrigel, but I used to do these types of invasion/migration experiments by coating the transwell insert with chemo attractants, such as ICAM-1 for T-cells, then adding the cells to the top of the transwell insert. You can also add a know activator of your cell type to the media that is known to promote invasion. If the cells still do not invade very well, you can start adjusting the media or trying different techniques with the matrigel.
Hope this helps!
As you said, your matrigel concentration might be high, another thing is the volume of matrigel. You have used 100 ul. If you are using a 24 well or a 12 well plate...try reducing the volume to 50 ul; the matrigel coating will be thin. Another point is serum concentration gradient. You have mentioned that you have used complete media in the lower chamber, you can try with 20% FBS. In the upper chamber media you can try with reduced serum content (0.5% FBS). All the best!!
Thank you for giving such a complete description of your experimental system. That helps a lot and I'm sure will provide you with lots of good suggestions.
1. PANC-1 cells do not require matrigel for attachment or growth. Are you using matrigel because the PET membrane is not tissue culture treated or to simulate ECM? If possible, try some experiments without the Matrigel.
2. As you suggested, you should also try using DMEM with 1% or 10% FBS in the upper chamber during the attachment phase. After attachment is complete, set up the chambers with 1% FBS in the top chamber and 10% in the bottom chamber. This will still provide a substantial gradient and the serum in the top chamber will prevent the cells from becoming too adherent to move. Also, 24 hours in SFM may cause the PANC cells to become sessile.
3. As mentioned above, the Matrigel layer is too thick or too dense. Try reducing the volume of Matrigel added to the top well or diluting it.
4. 24 hours for the migration part of the assay is probably not too long. You can check to see if the cells have migrated through the membrane and dropped off the bottom of the membrane by checking the culture medium in the bottom well for floating cells and by looking for adherent PANC cells attached to the plate in the bottom well with an inverted scope. In my experience, PANC cells are extremely slow growing with a doubling time of about 50 hours. This slow growth may indicate that the cells also are very slow migrating.
5. Maybe the pore size of the membrane is too small. If you can, try a larger pore size.
Good luck!
hello! you can do one thing . make the concentration of matrigel 0.5 mg/ml. Just coat the inner membrane with 40 ul and then without touching the membrane carefully spread the gel with pipette tip.
Secondly,
Cell density should be 5X10^5 cells/ml.
third after your intended time change the method of staining.Use H&E staining instead and dip the membrane in xylene and immediately proceed for counting
After the matrigel has gelled, there will be a liquid unbound portion sitting on top. Very very carefully aspirate off the unbound liquid, by tipping the invasion chamber horizontally and use a vacuum with a glass pipette to suck off the liquid. This needs to be done very carefully. If you leave the unbound liquid there you will not have very many cells that can invade.
Additionally, you can make your crystal violet in methanol to combine the fixing and staining step.
Your first problem is your choice of chemoattractant. 10% FCS, while commonly used, is one of the worst chemoattractants, especially for pancreatic cells. In our experience, pancreatic carcinoma cells reduce their migration and invasion toward FCS compared to BSA alone. This is why many have to use a 24hr invasion assay while in our conditions we use only a 4-6 hr invasion assay. I would recommend either 5ng/ml EGF or 10 or 50ng/ml HGF. When you use purified growth factors, you will need to coat the bottom of the transwell with laminin or another matrix so the cells will adhere.
Secondly, pancreatic cells invade better in collagen gels. And never use suction on a transwell that has been coated with matrix. Use a pipette instead to make sure you don't suction off the matrix (which would prevent the cells from adhering). We use the 8um pore size regularly, so that pore size is fine.
If you would like our protocol, just send me an e-mail at [email protected] and I'll send you it.
Good luck!
Dear all,
Thank you for the great suggestions given. I am really appreciate all these. I have successfully to get more cell invaded through the matrigel-coated membrane by modifying the following:
1) Coat the inner membrane of cell culture insert with 0.3 mg/ml (load 100 ul).
2) Gelling for 5 h in 37oC 5% CO2 incubator.
3) After 5 h gelling, discard carefully the unbound liquid (as mentioned by Prof. Kathleen) and rehydrate with SFM for 1 h.
4) Prepare cell suspension in DMEM containing 1% FBS.
5) Add 1 x 10^5 cells into upper chamber in 200 ul. While lower chamber loaded with SFM or 10% FBS as chemoattractant.
6) Incubate for 20 h.
A few reminders that I would like to mention:
1) The doubling time for PANC-1 cells is varied (as I checked online), some claimed that their doubling time should be around 30-40 h while others also claimed that should be around 50 h (as mentioned by Dr. Robert ).
2) Yes, PANC-1 is a slow growing cells so 20 h or 24 h incubation should be fine. I am not dare to go higher than that since the doubling times claimed by many are at least 30 h to the highest of 50 h.
Thanks again!
Dear Yuan-Seng,
All suggestions are fine and sure you will have better invasion with all changing of the protocol. You can try also higher concentration of the cells in upper chamber.
Good luck!
All such experiences are nice. You can try with this protocol. I hope that helps
Use MIVO by React4life! Add fluid-dynamic circulation to transwell....
- Badges
- Science method