Hi, is there any accurate protocol for single cell seeding? please suggest.
Count the cells. Dilute the volume by serial dilution to get 100 cells in 1ml.For ex if you get1 million count resuspend in 10 ml. Take 100 ul from 1st dilution and make 1ml. Take 10 ul from dilution 2 and make them to 1 ml. you get 100 cells in 1ml. Distribute100 ul per well.Distribute in 96 well plate. 80% well can have single cells.some wells have no cells. some wells may have 2 cells.omit the wells with no cells and more than 1 cell. In serial dilution ,mix well otherwise cells get settle in bottom
I clone cells as follows and works for me
I do 2 dilutions of cells:
first one: 10 cell/100 uL (1 mL total volumen)
second one: 0.5 cell/100 uL (10 mL total volumen)
With the first dilution, I seed the first row of wells, this is just to guarantee no losing cells (for example if you transduce with a lentiviral vector), because there are some cells that poorly survive when are cloned (you can also use conditioned media to improve survival)
with the second dilution i seed the rest of the plate. Of course, there will be a lot of empty wells (half of them), but it´s very rare to find more than one cell in any well.
check each well the other day after cloning and mark the ones with single cells
What about using the MMI CellEctor: You can visually select single cells from one well and deposit them into any other well. Watch the video to see how this works:
https://www.youtube.com/watch?v=vH_hFVYJL64
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