Migration expt with Transwell plates - (Mar/02/2007 )
Hi guys,
I'm doing a cell invasion/migration experiment. Placing transfected cells onto a membrane coated with Matrigel and allowing them to "invade" the membrane. Ultimately they get stained so that you can count the number of cells that "invaded". The problem I am running into is I am unable to reliably count the stained cells. The area I am counting is larger than the area my microscope provides so I am continuously moving the plate but unable to reliably tell what I've counted and not counted once I've moved it.
I tried drawing a grid on the bottom of the plate... no good.
I tried drawing a grid on a transparency, placing that under the plate but again I'm unable to see it once I get my cells in focus.
I have over 21 wells to count but without reliable numbers this experiment is futile.
If you have a suggestion or experience in this please I'm begging you to post!
Thanks
Amelia
Hi!
I've done that sort of assays, but I use 12-well plates and I don't count the whole well. To count migrating cells, I just count 5 fields of the well: if you see the well as a clock, I count fields in 12, 3, 6 and 9 and the center. Then I have 5 counted fields per well, none of them repeated and with enough data to do statistical analyses.
I hope it helps.
Good luck!
I've done that sort of assays, but I use 12-well plates and I don't count the whole well. To count migrating cells, I just count 5 fields of the well: if you see the well as a clock, I count fields in 12, 3, 6 and 9 and the center. Then I have 5 counted fields per well, none of them repeated and with enough data to do statistical analyses.
I hope it helps.
Good luck!
beyond counting you can stain fixed cells f.i. SRB or Alamar Blue
I've done that sort of assays, but I use 12-well plates and I don't count the whole well. To count migrating cells, I just count 5 fields of the well: if you see the well as a clock, I count fields in 12, 3, 6 and 9 and the center. Then I have 5 counted fields per well, none of them repeated and with enough data to do statistical analyses.
When I did experiment concerning estimation of invasive activity of primary tumor cell lines I estimate the invasion percent by counting cells in 5 randomize fields. firstly you should stain your invaded cells. The Most popular dye is Diff-Quik stain. After staining your inserts remove non-invasive cell from the other side of membrane with cotton stick. if you early see that invaded cells cover membrane uniformly ( check under microscope) than i put with thin marker points in five randomly places on the surface where you initially seed your cells. Then under mocroscop you count stained cells in this five marker pointed area. And so make with control insert. Make experiment in minimum three parallel. So you obtain 15 data points for experimental inserts and 15 - for control. It will be enough for statistical significance.
Thanks so much for your replies!
This will make my eyes and head feel so much better 