What are the uses for positive and negative controls in flow cytometry? - (Jan/04/2016 )

Hello everyone,

 

Sorry if this is a beginner's question or that it has been asked (and answered) before, but I would just like to know: what is the use of a positive control (eg: constitutively-expressing GFP) and a negative control (eg: no fluorophore or constitutively-expressing RFP - irrelevant fluorophore) in flow cytometry?

 

Background: I'm a Master student who just started some experiments with flow cytometry. My sample is a bacterium (Bacillus subtilis) that expresses GFP when induced by an extracellular stimulus. I have positive and negative controls (as described above) using the same bacterium and I'm wondering, what's the use of these controls in flow cytometry?

 

Correct me if I'm wrong, but negative control (no fluorophore or with irrelevant fluorophore) is to show the background auto-fluoroscence and also show where is the population of cells in my dot-plot so that I can gate only my cells while ignoring other pieces of background stuff (eg dust). Positive control is to show my cell population as well? Because I'm only using one color for detection in my experiment, the positive control cannot be for compensation reasons, right?

 

So can anyone tell me the uses of these controls?

 

Thank you,

ink12

Mostly correct.

 

The positive is to show that the equipment is working as you expect, such that you can detect a population of cells that do show fluorescence... if your +ve control wasn't working, how would you deduce what was a positive signal?

Thanks for the reply, bob1!

 

So technically, even without the positive and negative controls, I can still deduce which of the populations are my cells by looking at the top right-most corner of the dot-plot, right? And if I gate these cells and the histogram shows fluorescence, then they should positively be my cells then, especially if I gate the same populations of different samples under different extracellular stimuli to show the difference in fluorescence. So then, even with this method of deduction, are the controls still needed?

Controls are always needed - never skip the controls, your data will at best be suspect, and at worst be wrong!

 

You can't deduce anything from the position of the dot-plot and fluorescence - how do you account for auto-fluorescence? You can only deduce your cells in comparison to controls.

Right, thanks for the explanation bob1!

Hi 

I am trying to analyse Flow Data for receptor expression, but a little confused about the controls. I believe they were all from tubes with equal amounts of cells/mL stained and a couple tubes unstained. I am using skin cell lines to test for the expression of TLR. I also have a known positive which does express the receptor.

The controls are the confusing part...

 

1.) I have been given a set of Flow Cytometry data which has:

IC-PE (isotype control-pe), IC-FITC (isotype control FITC), ICAM-1and a couple of unstained tubes.

 

From my understanding the unstained tubes work as my background/negative

I also been advised that my IC-FITC is also the background/negative

 

2.)But here is the confusing part, what is the IC-PE?

Is there a difference between IC-PE and IC-FITC? Are they both the background, treated as the negative?

 

3.)What does the ICAM-1 represent, would this also be my positive control?

I also have other stained tubes which does express positive for the receptors as mentioned before, but wondered why I have been given ICAM-1 as well?

 

4.)Also regards to MFI, which is best to calculate for MFI?

I have been using the median values of the IC-FITC, unstained and the tubes with the receptor staining to compare differences using an unpaired t-test, but have been advised this is incorrect.

 

Any clarification would be greatly appreciated please. Thank you.

I'm not completely sure, as it would depend on the exact experimental setup but:

 

2) IC-PE would be a negative control for PE labeled samples - this provides the background signal for this particular stain using an isotype (non-specific from the same species) antibody conjugated to PE. Both the IC-PE and IC-FITC are negatives for the particular stains.

 

3) ICAM-1 is probably the test, or it could be a positive control - hard to say without further information

 

4)MFI would generally be measured as the change in fluorescence intensity over a control - as a relative expression level measure. You would generally use the median fluorescence intensity from a sample compared to the control.