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# seeding of cells for transfection

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### #1 seasons

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Posted 14 March 2005 - 03:18 PM

is my protocol wrong or right?

1. dilute your cells to give 13ml cell suspension containing 130000cells/ml.

2. add 2ml medium to each well in two 6-well plates.

3. seed out 1.3x10^5 cells in each well of the 6-well plates.

if each well should have 1.3x10^5 cells, then why should i add 2ml medium in it, since the cells would be diluted. is step 2 wrong or right?

thank you.

### #2 pcrman

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Posted 14 March 2005 - 04:30 PM

If you diluted concentration is 1.3x10^5, and add 2 ml into each well, then there will be 2.6x10^5 cells/well.  You need add another 13 ml medium to the dilution, and then 2ml will give you exact 1.3x10^5 cells/well.

I think step 2 means adding the diluted cells to the plate and step 3 just explains what you did in step 2.

### #3 fred_33

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Posted 15 March 2005 - 03:03 AM

hi
i think that in step 2 you add a cell free medium
and in step 3 you seed your cells
the final volume in each well is 3ml and that is ok
The step 2 ensure a good repartition of you cells when you seed the plate.

But if 2ml/well is ok for your cells, pcrman is right.

Edited by fred_33, 15 March 2005 - 03:03 AM.

### #4 seasons

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Posted 17 March 2005 - 03:37 AM

fred_33, on Mar 15 2005, 04:03 AM, said:

hi
i think that in step 2 you add a cell free medium
and in step 3 you seed your cells
the final volume in each well is 3ml and that is ok
The step 2 ensure a good repartition of you cells when you seed the plate.

But if 2ml/well is ok for your cells, pcrman is right.
So you meant  that in step 3 i add 1 ml of my cell suspension with 130000cells/ml. When i add this 1ml,  the cell concentration won't be 130000cells/ml. since i already have 2ml of cell free medium. How many cells do you usually have in each well?

### #5 fred_33

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Posted 17 March 2005 - 03:49 AM

hi
the number of cells won't change if you further add 2ml of cell free medium and that's the important point.
for note, i usually seed 150 000 to 300 000 cells in a 6 well-plate.