2 replies to this topic

[icome]

In the invasion assay, I coated the matrigel on transwells in a 24-well dish. The method I used is to dilute matrigel in ddWater and  to add it on the filters followed by air-drying. The problem is that the gel cannot be smoothly paved on a filter after drying. The gel tends to form a "basin" that has a lower level at the center but a higher level around. This is because water tends to adhere to the wall of transwells. The basin would let cells concentrate at the center rather than spreading evenly during invasion. How could I pave the gel smoothly?
My boss suggested that adding something such as water on the gel during air-drying may force the surface of the gel to be smooth. Is this able to be done? Does anyone have better idea? Thanks for your reply!

Edited by icome, 18 October 2009 - 09:18 PM.

[amelodie]

This is the protocol we are using in the lab: matrigel is diluted 1:1 in IMDM, aliquoted and stored at -20C.
Aliquots are thawed overnight on ice before use. Before coating, we are storing the plates/filters on ice to have them ice-cold. Then we are just adding the matrigel, removing the excess and leave the plates flat and untouched on ice for at least 15 min. After that, just pipette the excess of matrigel if any, let rest 5 more min on ice, 5 min at RT and use.
Never had problem like that, hope it will work for you,

[icome]

Excuse me! I have a question. You mean that I should jsut add the matrigel on the filter followed by removing it. Could the matrigel be coated on the filter by this method? I am afraid of all the matrigel being removed from the filte! Would there be an enough amount of matrigel remaining on the filter? Thanks for reply!

amelodie, on Oct 22 2009, 05:08 AM, said:

This is the protocol we are using in the lab: matrigel is diluted 1:1 in IMDM, aliquoted and stored at -20C.
Aliquots are thawed overnight on ice before use. Before coating, we are storing the plates/filters on ice to have them ice-cold. Then we are just adding the matrigel, removing the excess and leave the plates flat and untouched on ice for at least 15 min. After that, just pipette the excess of matrigel if any, let rest 5 more min on ice, 5 min at RT and use.
Never had problem like that, hope it will work for you,

Edited by icome, 25 October 2009 - 09:23 PM.