My Kg1a cell lines grow very well in flask, but when I seed it in 96 wells plate give only 25 % viability
Hello Haya Fahad
You need to consider the following when you plate cells in 96-well plate.
1. It is important to ensure that the plate is level. Place the 96-well plate on a flat surface and use a spirit level to ensure the plate is level. This will help ensure even distribution of cells throughout the plate.
2. After completing seeding your plate, leave the plate in the hood for 1 hour after seeding. This would be necessary because the moment you place the plate in the incubator it heats up rapidly to 37 degree C, and this causes the media to be affected by rough thermal currents that causes your seeding cells to settle unevenly. Just tap the sides of a 96 well plate to ensure the cells are evenly distributed throughout the well.
3. Ensure that the volume is consistent and prevent air bubble formation during cell seeding.
4. The edge effect is a common problem in 96-well plate. Increased evaporation from the peripheral wells is assumed to be associated with this edge effect phenomenon. Temperature differences across the plate and evaporation effects in the edge wells during incubation have been described as two possible influencing factors for irregular patterns of cell growth and distribution especially in the plate periphery. It is assumed that evaporation in the outer wells may lead to an accumulation of medium components (like salts), thereby affecting cell metabolism.
One way to circumvent the edge effect is to avoid seeding cells in the peripheral wells or add media or sterile water in these edge wells. Another approach would be to minimize the impact of temperature gradient by pre-incubating the plate with freshly seeded cells for 1–2 hours at room temperature which results in a more even cell distribution and adhesion pattern. This procedure might be critical depending on the cell type used with regard to cell viability. The creation of a constant micro-environment by reducing evaporation to a minimum and achieving an almost consistent humidity and temperature level would be necessary.
You may want to refer to the paper attached below.
https://journals.sagepub.com/doi/pdf/10.1177/1087057103256465#:~:text=Pre%2Dincubation%20of%20plates%20with,effect%20in%20cell%2Dbased%20assays.
Best.
I seed 80ul of cell suspension in each well (1000cell/ml )
do you think is good volume
I attached my calculations
Malcolm Nobre
Hello Haya,
You should seed cells in a minimum volume of 100ul per well in a 96 well plate.
Suppose you want to seed 1000 cells per 100ul per well, then for 100 wells (always consider extra wells), you will require
1000 x 100 = 100,000 cells in 10000ul (10ml).
Then from 10ml cell suspension, you pipette out 100ul per well which will give you 1000 cells/100ul/well. Always pipette the cell suspension up and down a number of times so that each well will receive equal number of cells.
If you need to seed just one well, then you make a cell suspension for 5 wells (4 wells extra) because pipetting small volumes can lead to pipetting error. So to avoid this error you should increase the volume. So for 5 wells, you will require 5000 cells in 500ul. Then pipette the cell suspension up and down and add 100ul to one well.
Best.
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