When seeding C2C12 cells in 384-well plate at a density of 50 per well and continuing to culture for 48 hours, there is always a randomly empty area, and cells tend to grow more at the edges of the well.
What methods and techniques can be used to achieve more even seeding? Thanks!
Hello Claire Hong Kong SAR Zhong
Suggestions that may help to achieve even seeding.
1. Transfer the cell suspension to a trough for easy access by a multichannel pipette. Alternatively, you may divide the cell suspension evenly into microcentrifuge tubes that can be used with a multichannel pipette, providing enough volume to seed the cells with 25µL addition per well.
2. Make sure that you mix the cell suspension thoroughly before any addition to ensure even distribution of cells. Dispense the cells directly in the middle of the well.
3. Cell culture media tend to foam due to the high protein content of supplemented serum. Air bubbles can hinder cell attachment during seeding and therefore create well-to-well variations of cell numbers. Especially in smaller plate formats like 384-well plates, air bubbles can have a big impact due to the small growth area per well. So, although mixing is necessary, you should not overdo it and avoid too much pipetting to reduce air bubble formation. Gentle pipetting also reduces shear forces and stress on your cells.
4. To achieve an equal cell distribution, I use the figure-eight movement, while others may prefer a cross-like movement of the plate. Both techniques lead to a better distribution of cells. However, the smaller the vessel diameter like the 384-well plate, the less movement of the liquid you have, leading to an uneven distribution of cells. To circumvent this effect, during cell seeding, first dilute the cell suspension to the desired concentration in a tube and then pipette the final volume to the culture vessel in one step.
5. After seeding cells on the plate, leave it to rest in the biosafety cabinet for 1 hour at room temperature. 384-well plates are sensitive to thermal gradients, which can cause edge effects.
6. When 384-well plates are kept in tall stacks, it will take longer to equilibrate to the surrounding temperature than plates which are arranged separately. Inefficient temperature equilibration can result in temperature gradient effects between wells in the center and wells at the edges of a plate. Plate stacking may exacerbate such cross-plate temperature gradients as well as causing differential gradient patterns between plates within the same stack. This may affect assay performance and cell migration towards the edge of the wells when seeding.
Best.
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