Does anyone have experience doing scratch assays in stem cells in 96 well plate using the wound maker? I notice that after doing the scratch and washing twice, the cells round up and there are gaps between cells although were confluent at baseline?
Wells are coated with matrigel.
Dear Nihad
To my experience if the cell confluency was above 90% that is perfect to start with scratch assay. However, as you mentioned you are scratching cells in 96 well plate- I presume that you are scratching many wells at one time. As far as rounding of your cells is concerened, it happens while washing with PBS right?
if I would be doing such experiment, I would prefer using 1x DPBS which contains Ca+ and Mgcl2- this will not let your cells detach off the surface. Alternatively I would wash my cells with the media they are grown in a couple of times to remove scratch debris. I hope this helps
Dear Nihad Abouelazm,
I do not have experience Stem cells but do a lot for primary skin cells especially big challenge for epithelial skin cell. I guess you need to understand your cell behaviour and suitable confluence prior to the scratch procedure. I rather use 48 well-plate compared to 96-well plate as the space too small and could provide high variation of scratching for each well. You also need to control the medium or buffer temperature as it could influence the native behaviour of your cells. Best of luck!
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