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Indirect Flowcytometry


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22 replies to this topic

#1 baaran

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Posted 21 June 2011 - 05:41 AM

Hi all
I am new in Flowcytometry. In my thesis I am to assess the binding ability of an scFv antibody to the surface of a targeted cell line. For this purpose, after harvesting and washing the cells, I first incubate them with the scFv antibody on ice for 2 hrs, then (After Washing) anti-fd antibody will be added to the cells and incubate for 30 min at RT. Then (After Washing) the conjugated antibody will be added for 30 min at RT. You know this procedure needs to be set up but I don't know how I can change the protocol. I use 1 million cells for each condition, 1000 scFv per cell, anti-fd with the final concentration of 100 microliter/ml and conjugated antibody at the final concentration of 100 microliter/ml. Which part can I change? your urgent response is highly appreciated.

#2 Rnotk

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Posted 26 June 2011 - 12:03 PM

I am not sure what you are trying to do?? what is the third "conjugeted" antibody against?
Also, why you are trying to modify this protocol? do you have any particular reason that you need to modify??
FACS staining is similar to immunohistochemistry, if you want to change, you can change any part of protocol, but need reason.

#3 baaran

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Posted 03 July 2011 - 06:23 AM

I am not sure what you are trying to do?? what is the third "conjugeted" antibody against?
Also, why you are trying to modify this protocol? do you have any particular reason that you need to modify??
FACS staining is similar to immunohistochemistry, if you want to change, you can change any part of protocol, but need reason.


Thanks for your response,
You know I am trying to assess the binding ability of a single chain antibody(scFv) to the surface of a cell line. This scFv is expressed on the surface of M13 phage. So for my purpose I should
1. treat the cells with the scFv
2. Use the anti-fd antibody( an antibody that binds to the fd or M13 phages). This antibody is from the IgG class
3. Apply the fluorescent (FITC)conjugated antibody that binds to the IgG
Also I treat the same amount of cells(1 million) with M13 phage as the Isotype control to exclude the non-specific binding of the M13 phage to the cell surface
But the problem is that it didn't work and I've not been able to get any result :(
I know that I should set up concentrations of the anti-fd and FITC conjugated antibodies BUT how?
Attached is the product information of the Sigma for using anti-fd.

I don't have much time...Please help me out...

Attached Files



#4 Rnotk

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Posted 05 July 2011 - 01:22 PM

what do you mean by "it didnt work" or "you are not getting any result"?
Not sure what kind of problem you are having, so it is hard to comment on that.

#5 baaran

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Posted 05 July 2011 - 10:12 PM

what do you mean by "it didnt work" or "you are not getting any result"?
Not sure what kind of problem you are having, so it is hard to comment on that.


I use M13 phage as the isotype control in my experiment. I expect that my test conditions show more fluorescence than Isotype control. But in all my experiments, fluorescence emission from the Isotype was equal or more than my test tubes.

#6 Rnotk

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Posted 09 July 2011 - 11:47 AM

if I am still misunderstanding your experiment, sorry
but I thought you said your antibody anti-fd binds to M13 phage, so is it suppose to have more fluorescent if you use M13 phage??

#7 baaran

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Posted 11 July 2011 - 01:19 AM

if I am still misunderstanding your experiment, sorry
but I thought you said your antibody anti-fd binds to M13 phage, so is it suppose to have more fluorescent if you use M13 phage??



You know I am working with scFv antibodies, expressed on the surface of a phagemid( based on M13 bacteriophage). These phages can bind to the target antigen via their specific inserted scFv antibody. On the other hand,an antibody(i.e. anti-fd antibody ) against structural proteins of the phage, can bind to the phages. Afterward we can use a conjugated antibody to detect the anti-fd antibody.
Something like this:
Antigen expressed on the cell surface ---Phage with specific antibody against the targeted antigen ---anti-fd antibody --- Conjugated antibody.

#8 Rnotk

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Posted 11 July 2011 - 08:09 PM

OK I think I understand the system that you are trying
so your isotype (M13) does not express svFv, so it suppose not to bind to the target protein??

BYW, you are using anti-IgG conjugeted antibody as tertiary, this does not bind to the target cell?

#9 baaran

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Posted 13 July 2011 - 01:35 AM

OK I think I understand the system that you are trying
so your isotype (M13) does not express svFv, so it suppose not to bind to the target protein??

BYW, you are using anti-IgG conjugeted antibody as tertiary, this does not bind to the target cell?

You are completely right now. The tertiary conjugated antibody just binds to the anti-fd (because the anti-fd is class 1 IgG). And we suppose that anti-fd will bind to the specific phages bound to the target antigen via their scFv antibody.

Edited by baaran, 13 July 2011 - 03:02 AM.


#10 Rnotk

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Posted 13 July 2011 - 02:16 PM

have you ever done control without tertiary antibody? (to see autofluorescent)

How is the cells looks like? after all the staining procedure?

Did you fix the target cells before staining? or any of your procedure permeablize the target cell?

#11 baaran

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Posted 14 July 2011 - 01:58 AM

have you ever done control without tertiary antibody? (to see autofluorescent)

How is the cells looks like? after all the staining procedure?

Did you fix the target cells before staining? or any of your procedure permeablize the target cell?



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.

#12 baaran

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Posted 14 July 2011 - 02:13 AM

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?

#13 Rnotk

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Posted 14 July 2011 - 02:51 PM

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.

#14 Rnotk

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Posted 14 July 2011 - 02:55 PM

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

#15 baaran

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Posted 15 July 2011 - 03:49 AM

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...




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