Force Detachment Assay

Force Detachment Assay
Prepare the desired number of 24-well plates by shaving-off the edge of each plate until
smooth, in order for a plate cover to have a snug-fit. Also, pre-cut a thin pad to fit on the
inside of the plate-cover.
Day 1
1. Coat 24-well plates (middle 16 wells) with concentrations of collagen and/or
fibronectin (300ul coats each well) for 24 hrs. (make sure to have two wells of the
same concentration)
Day 2
Wash thoroughly all wells with saline without disrupting the matrix.
Block the wells with 2% BSA in saline for 1 hr at 37C.
Wash again with saline.
Plate cells at desired density (with YCF 3x103; OCF 5x103) for 24 hrs.
Day 3
6. Fill all wells with regular feeding media up to the brim of each well.
7. Slowly and gently, start at one end of the plate by applying the pressure sensitive
film (Becton Dickinson, cat. # 35-3073).
8. As applying film: keep the loose-end taut and slowly move a straight -edge (i.e. a
pencil, or a wooden block) across the plate to press down on the adhesive film.
9. Invert the plates to be spun and place inside the centrifuge. Note: have another
set of control plates inverted on the bench (at least one control plate per
experimental day).
10. Select the desired force (i.e. 600 x g, 800 x g, etc.), spin for 5 min.
11. After plates have been spun, keep inverted until ready to use.
12. Invert plates, suction off media.
13. Add trypsin (300ul) until cells detach (make sure to look for detachment under the
scope), add media (300ul to each well). Combine two wells of the same condition
into one centrifuge tube (1.5 ml eppendorf tubes) and spin down the cells at
800rpm for 8 min.
14. Make sure to look for a pellet of cells, have the pellet resuspend in 50ul of media
(since the pellet is very small).
15. Count cells.
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