0.1 million cells in 1 ml media were added per well. Same pattern observed in all 12 wells during multiple platings.
This is a common issue in multi-well plates. I recommend incubating the wells with media for 1hr prior to plating. This allows serum proteins to attach to the culture treated plastic to aid in quicker attachment. Additionally, after plating the cells you should vigorously shake the plate from side to side and up and down to ensure even distribution. Try not to disturb the plate while transferring it to the incubator and return 30min later to repeat shaking.
Hello Umang Tyagi
Uneven distribution of cells arises due to factors such as:
1) The macro scale turbulent flow of cell suspension as it is added to the well,
2) Disturbing the cell suspension as plates are moved to the incubator,
3) The meniscus that forms around the wall of the culture plate (this effect is more pronounced with smaller sized wells).
It is a common trend that after pipetting the cell suspension into the wells of the plate, most of them move the plate in a circular manner with the hope that the cells will get evenly distributed. This type of motion will actually cause the cells to come together in the center. To achieve an equal distribution in the well, you could either use a figure-eight movement or a cross like movement of the plate namely, side to side and then forward and backward.
Hope this helps!
You could possibly also allow your cells to attach for 30 min at RT, leaving the plate in the biosafety hood before placing the plate into the incubator.
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