I have seeded 10000 cells/100 ul media/well of a 96 well plate and want to treat them with 1, 2, 5, 10, 25, 50 & 100 ug of a protein extract, having concentration 1.8 ug/ul,to know the cytotoxic effect. Is it be ok to add 1, 2, 5...... ul of protein extract or I should calculate protein concentration for 100 ul as I'm seed total of 100 ul in each well.
100 ug of protein extract should be quite high concentration in this case when you have 1.8 ug/ul. It means, that you have to add at least 55.56 ul of your extract into one well to achieve 100 ug/well. And it means, that you will dilute your cell culture medium quiet a lot. Do you have in mind concentrations 100 ug/100 ul, 50ug/100 ul, etc. or something else? (100 ug/ml?)
It's up to your experimental design. If you aim to treat your cells with amount of 1, 2, 5... ug, then you have to make a calculation like this:
For instance to treat your cells with 1ug extract you should take 1/1,8 ul from stock extract or for 2 ug you should take 2/1,8 since your stock protein extract concentration is 1,8 ug/ul.
However if you aim to treat with concentration of 1ug/ul, 2ug/ul, ...ug/ul then yes you should calculate for 100ul but be aware that it is not possible to prepare more concentrated solution than 1,8 ug/ul.
100 ug of protein extract should be quite high concentration in this case when you have 1.8 ug/ul. It means, that you have to add at least 55.56 ul of your extract into one well to achieve 100 ug/well. And it means, that you will dilute your cell culture medium quiet a lot. Do you have in mind concentrations 100 ug/100 ul, 50ug/100 ul, etc. or something else? (100 ug/ml?)
You can still carry you experiment your way!!
I thing that you should make estimation in such way that your total volume (volume of cells + volume of protein extract) is equal to100 ul; corresponding to about 10000 cells/100 ul media/well.
For example: if the volume of cells is 80 ul, the volume of protein extract will be 20 ul for each of the treated mass (1, 2, 5, 10, 25, 50 & 100 ug ).
Then, you prepare your protein extract at different concentrations in such way way 20 ul will give the desired mass
eg
for 100 ug you take 20uL of protein extract 5 ug/ul
for 50 ug you take 20uL of protein extract 2.5 ug/ul
for 25 ug you take 20uL of protein extract 1.25 ug/ul
for 10 ug you take 20uL of protein extract 0.5 ug/ul
for 5 ug you take 20uL of protein extract 0.25 ug/ul
for 2 ug you take 20uL of protein extract 0.1 ug/ul
for 1 ug you take 20L of protein extract 0.05 ug/ul
If your protein concentration available is 1.8 ug/ul; if the volume of cells will be 44.5 ul, the volume of protein extract will be 55.5 ul for each of the treated mass; but the calculations will be little fastidious
We seeded lymphocytes @ 120 micro lit of medium containing 33000 cells/Ml per well for MTT assay of cytotoxity of tobacco extract after 4 hr incubation
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