Hello Mohan,

Before continue your experiments its need to clarify does this signal come from bacteria or not. Did you perform this check? Some Ab reagents tends to aggregate. Mix all, except bacteria, and run.

Some points should be considered here.

Nature of sample

Was acquisition done at a constant voltage for each sample .

Even though these two fluorochromes won't interfere with each have you compensated them.

In positive sample  and in the LL quadrant  two populations are seen so gating should be performed accordingly

Dear Mohan,

In your FACS analisis you used 2 antibodies, one connected with FITC and second with tandem PerCP-CyP5-5A . Have you performed analysis with one antibody only or with isotype control?

It is realy important because the positive data which you presented on dot plots may be autofluorescence of bacterial cells.

Best regards,

Sławek

Dear Mohan

In the same way you have to :

-Perform the compensation

-Acquire your positive and negative bacteria alone (without antibodies) to determine autofluorescence in your samples.

When I look at your results I am pretty sure that you have to do compensations.

Best regards

Hi, 

May I know how many fluorochromes you've used to stain ?

Dear All,

Thank you very much for all the suggestions. I will try to clarify the questions for a clearer image. 

I used FITC (530/30) and PerCP-Cy5-5 (670 LP). I didn"t perform compensation in ths test because I obtained 0 in the previous test.

I have done the single controls and the file is attached herewith. My main focus is FITC-only sample. Because that sample provided + results with the - sample also. 

All the experiments are done in constant voltage. I adjusted the voltage using unstained sample and maintained it throughout. 

I am unable to detect 2 populations in the LL in the positive sample. If you can just mark it, it would be of great help.

Thanks.

Hi Mohan

You have to perform compensation. In the single stain tube FITC we clearly see an overlap of this fluorescence in the PerCP Cy5.5. 

But you will have FITC positive events for negative bacteria (I think° and i don?t know why.

Best regards

Dear Mathieu,

Thanks for the advice.  I just tried that using the Flojo and the both positive fraction changed from 2.94 to 2.72 in the + bacteria sample. 

Is there a probability of the specifity of the antibodies used for this experiment? 

Thanks 

Hello

Look at the papers here , Hope you find explanation for your questions

Good Luck

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC100149/

http://www.ncbi.nlm.nih.gov/pubmed/15711926

Try to Compensate FITC using flowjo. Use samples which have clear positive and negative population for compensation. this might improve your data as dual positive population is  appearing as trail.

Dear Asif,

I did the compensation already. But the percentages are very less because the emission spectrums are far from each other. Do u think the boundary should be around from 200 instead of the point I selected? I will attach the compensated population for your reference.

Hello Mohan,

Before continue your experiments its need to clarify does this signal come from bacteria or not. Did you perform this check? Some Ab reagents tends to aggregate. Mix all, except bacteria, and run.

Dear Daniil, I will consider that next time.

Thanks

Are your bacteria photosynthetic?  Pigments may contribute to autofluorescence.  

Dear Jean,

This is not a photosynthetic bacteria.

Thanks

Dear Mohan,

It is a wondering thing when we find the evidences that some of the bacteria contributes autofluorescence, because of its physical nature or may be due to some of its environmental cause. Interestingly, those bacteria almost falls at the FITC region. (Eg. Cyanobacteria, E.coli & so on) (Ref. Research article  attached below).

In your case, you are sure which are antigen +tive and Ag -tive from western blotting andELISA. But, while staining with FACS markers to your sample, you are getting FITC positive for your Ag +tive bacteria which is expected. Unfortunately, you founnd same FITC positive for your Ag -tive bacteria.

Here I doubt, why not the Ag -tive bacteria contribute autoflorescence, since the sample collected from environmental source where we can see autofluorescence contributing bateria...?

If so, it is sure that your Ag -tive bacteria contributes autofluorescence and falls at the region of FITC and will not have the problem with spectral overlap between FITC and PerCP-Cy5.5.

So, if my assumption is correct, then I can suggest you to use the marker of interest tagging with some other fluorophore and its up to you.

That's all about my point of view.

Best,

Kadar Moideen Abbas

Dear Kader,

Thank you very much for your suggestion. But I have to clarify one thing here. The - bacteria i used is a staphylococcus strain taken from the ATCC. That is why I was wandering. 

But I will consider the suggestion that changing fluorophore will provide me any new insights.

Thanks

Like Kader described very well; expecially bacteria or also cells like macrophages it is always difficult to work with fitc or even pe conjugated antibodies; if your intrument allows it, go for BV dyes (e.g. BV421) or Alexa647...

cheers

Dear Kadar,

I am wondering why your negative and positive sample looked like containing different populations?  Also how did you prepare your negative control?  Using a isotype FITC control antibody?  Did you use the same antibody for your Flow and your WB?  Just make sure that it works for Flow though. Another option is to use far red labeling to avoid autofluorescence issue. 

chees,

Dear Jochen,

I am using bacterial red stain by molecular probes to analyze the bacterial population in PerCP-Cy5.5. FITC-antibody is used to detect a specific antigen present in the bacteria. 

Dear Liqun,

I didn't get your question correctly. Which are the different populations you explain? I used an antigen-positive bacteria and antigen-negative bacteria. Negative control is the antigen negative bacteria. So I did double staining for 1. bacteria and 2. antigens. My problem is antigen-negative bacteria is also stained by FITC-antigen specific antibody. WB and FC both done using same antibody except that the FC is done using FITC- conjugate. WB was done using HRP-conjugate secondary antibody. No isotype control is used. 

Thanks,

Dear Mohan

1)  I am wondering how you get your +bacteria?  Did you make them by introducing the antigen to a colony of  –bacteria purchased from ATCC?

2)  If you used PerCP-Cy5.5 to label bacteria, why you have different labeling profiles between your – and + bacteria, if the only difference of your – and + bacteria is one containing antigen and the other does not?  

3)  Have you checked your –bacteria and +bacteria with only PerCP-Cy5.5 staining, respectively, under the same setting and plot you showed here? If you did and still saw signal in FITC channel for both of them, then the signal is very likely an autofluoresence.

4)  If you conjugated FITC after you incubated the primary antibody with bacteria, you may want to run a FITC secondary antibody only control with your -bacteria.  

If everything works so far, you should be able to show your +bacteria with FITC labeling on Flow, otherwise, your primary antibody did not work here.

As I mentioned before, antibody works on WB does not guarantee it works for Flow for deferent reasons. 

cheers