4 replies to this topic

[brand]

Dears,

it would be grateful to know how can i calculate the antibody dilution needs to stain given number of cells rather than the volume of the solution.

That is I need i micro gram of antibody( 1mg/ml stock) to stain 1 million cells

[bob1]

There is no defined cell:antibody ratio, it is always done by volume!

if you have 1 mg/ml and you need to get 1 ug use volume = amount/concentration.  Remember to keep your units consistent.

[leelee]

bob1, on 23 February 2012 - 12:19 PM, said:

There is no defined cell:antibody ratio, it is always done by volume!

Have to disagree with you there. The antibodies I use come with a recommended amount of antibody to use per (for example) million cells, and not a working concentration.

In my opinion the volume you stain in is less important than the amount of antibody available to stain your cells. It is far more consistent to use the same ratio of antibody to cells per experiment than to use a defined concentration.
Of course, if you ALWAYS have the same number of cells, its probably not going to be a problem.

In your example, brand, this is how I would do it:

Your stock is 1mg/ml which is 1ug/ul- so add 1ul (1ug) of your antibody to your 1 million cells, in your desired volume. I probably would do 200ul, but that's because I do all of my staining in plates.

In any case, I would recommend titrating your antibody out.

[brand]

Dear leelee and bob 1

thank you for the answer.Method by leelee is the best practice in this scenario.

[bob1]

What I was referring to is that for each antibody the ratio of cells to antibody will be different, hence most antibody applications are done based on a dilution ratio rather than an absolute cell number.

I think of it this way - I could easily put 1 million cells into 1 ml  and add 1 ug, but I could also have 1 million cells in 100 ml, for which 1 ug would be too small an amount to stain anything effectively...