How to seed less number of cells - (Jul/03/2008 )
I have a quick question - i want to know how to seed less cells , whenever i i need to plate 500 or 600 cells i always have a problem as my original sample has more cells so how do i dilute to get the cell number i need. Please explain in detail. Would be great. Thanks:)
If you are needing to seed a specific number of cells you must count your cells before seeding. After you trypsinize and resuspend the cells in media, you take about 30-40ul and use a hemacytometer to count the number of cells per ml. A hemacytometer is a special slide which any lab that does tissue culture should have. Ask in your lab if you have one and have someone show you how to use it. It's hard to explain in a post but there are also video demonstrations that show you how to use this. www.jove.com/index/details.stp?ID=752 for example. Just search the internet and you can find many of these. It's really easy and just requires you to count and do a bit of basic math. By counting your cells you can then determine the volume of the trypsinized/resuspended cells you need to add to your well/plate to seed the specific number of cells you want.
As suggested by rkay:
Count your cells using Haemoctyometer (0.1% Trypan blue added 1:1 to the small aliquot of cells to be counted, blue cells are dead).
Take the number of cells you have and divide it by the number of grids counted and multiply by your dilution factor (=2 if you added 1:1 as above).
Now you have the number of cells per 0.1ml, so add 104 to this number to get cells per ml(i.e if you worked out 39 cells, you have 39*104 cells/ml).
Use Vi*Ci=Vf*Cf where V and C stand for volume and concentration respectively and i and f stand for initial and final respectively, to calculate your dilution.
I counted 9 grids on one side of my haemocytometer with a 1:1 dilution of my cells with trypan blue and got 300 cells
300/9 = 33.333 (number of grids)
33.333*2 = 16.6665 cells per 0.1 ml (dilution factor)
= 16.6665 *104 cells per ml
I have 10 ml of cell suspension and need 1*104 cells per ml so...
Vi = 10
Ci = 16.6665*104
Cf = 1*104
so I would need to resuspend my cells in 166.65 ml to get 1*104 cells per ml.
Note that you could spin down and resuspend your cells in a smaller volume with an easy to calculate concentration and then take an aliquot and dilute that. Also Vi*Ci is your total cell number.