Indirect Flowcytometry - (Jun/21/2011 )

Rnotk on Wed Jul 13 22:16:24 2011 said:

I haven't done the control without tertiary antibody ... But i think it is not necessary.
Before the staining procedure, I mean when harvesting the cells, they look happy with viability more than 95%. All the procedure is done in flowcytometry tubes and I haven't seen them under microscope.
I don't use any permeabilize or fixation reagent. Because I thought these reagents might lead to the internalization of the surface antigens.

You know I think that the most important part to make any changes is the concentration of anti-fd antibody. But how? In the previous replies I've attached the data sheet of the anti-fd we are using in our experiments. What do you think? How can I make changes to the concentration of fd-antibody?

first of all, fixing cell prevent internalization of antibody. If you dont fix cell, and incubate cell (expecially at RT), antibody will be internalized.

for the concentration of antibody, you said you use 100ul/ml. is this mean you are using 1:10 dilution. If so it is too high.
It is better to use 1:1000 dilution as a starting point.

for your conjugated antibody, the good starting dilution is 1:1000-1:500
Anything more would be end up as high background.

1x10^6 cells/sample seems appropriate for Flow cytometry analysis

I am using approximately the same concentration of conjugated antibody and also 10^6 cells per sample, BUT how much anti-fd should I use?
You know I use 3 conditions with different numbers of scFv per cell i.e. First condition : 10^6 cells with 100 scFv per cell.
Second condition: 10^6 cells with 500 scFv per cell.
Third condition: 10^6 cells with 100 scFv per cell.
I don't know what proportion of anti-fd should be used...

If I decided to use for example 1:1000 dilution of anti-fd, then I will use same dilution for all three conditions. otherwise it is hard to interpretate.

Maybe you are right BUT based on data sheet of the anti-fd, the amount of anti-fd should be adjusted according to the number of scFvs. Also I've tried the same concentration of anti-fd for three conditions but it didn't work...

how about doing the experiment without M13 phage? (as negative control)
The problem you are having is the isotype gives you high fluorescent value right?

some antibody such as anti-fd or tertiary antibody somehow attaching regardless of scFv affinity.

I understand that you want to change the concentration of anti-fd according to the scFv concentration,
but if the system is working, M13 isotype should not give high value regardless of concentration of anti-fd antibody.

or simply you are using too high concentration of each antibody since you are using three antibodies and more antibodies will amplify signal quite significantly

I can't omit the M13 Isotype control because it is the backbone of my scFvs. I have to use it to show how much the M13 alone can bind to the target cells. "the amount of anti-fd should be adjusted according to the number of scFvs" BUT the scFvs are expressed at the surface of M13 bacteriophage. And also anti-fd is an antibody that binds to the M13 phages. So in order to exclude the nonspecific binding of the M13 phage(alone with no scFv on its surface), I must use the M13 phage alone.
And from your previous replies, i'd better to use fixative reagents before treating the cells with scFv?

so for your isotype you have M13 that binds to the target protein on the surface of target cell.
Does this M13 binds to the target cell using scFv on thier surface or different interaction?

I thought your isotype M13 suppose not to bind target cell.
if it does, what is your experimental? not phage but pulified scFv?

The reason that I suggest no M13 is to check the system, because you are having too high background
so to me you either using too high concentration of each antibody or antibody(ies) you are using bind unexpected mannar.