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What are the uses for positive and negative controls in flow cytometry?

positive negative controls flow cytometry

Best Answer bob1, 04 January 2016 - 12:14 PM

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?

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#1 ink12

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Posted 04 January 2016 - 09:43 AM

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



#2 bob1

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Posted 04 January 2016 - 12:14 PM   Best Answer

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?



#3 ink12

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Posted 04 January 2016 - 01:08 PM

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?



#4 bob1

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Posted 04 January 2016 - 03:11 PM

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.



#5 ink12

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Posted 05 January 2016 - 02:52 AM

Right, thanks for the explanation bob1!







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