So, I have been trying to quantify the migrated cells through transwells without using Matrigel (because I just want to quantify the natural migration rate of HEK 293T).
1. I plate them on the transwells and let them sit for 6-8 hrs, with complete media on both top and bottom of transwells.
2.Then, I aspirate old media on both top and bottom. I put 300 micro litre DMEM only on top of transwell and 300 micro litre of complete media on bottom chamber.
3. I keep it 24 hours undisturbed in the incubator for the migration to occur.
4. Then I aspirate all the old media, wash them with DPBS and then fixation and staining.
My concern is:
1. HEK 293T being weakly adherent comes of with DPBS wash.
2. Post staining some cells have weird morphology, so it becomes difficult to image and count them under BF.
Please give me suggestion to do this standardisation better.
If you have a problem with the cell counting and you are afraid of losing the cells before fixation, you can try another migration system. You may use the fluorescent dye (like DiO, Dil, Cell Tracker CMFDA, or other) and the Corning FluoroBlok inserts. In this case, you need a fluorometer to measure the fluorescent signal given by the cells that migrated to the bottom side of the membrane. The membrane in the insert does not pass the light so, the signal from the non-migrated cells is excluded and you read the plate without washing and in several timepoints. In this approach, you do not get the absolute number of cells, but the relative fluorescence unless you prepare a standard curve.
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