The unstained cells are used as a control when performing flow cytometry. These cells lie in the lower left quadrant of a biparametric histogram representation of cell population, when dual staining of cells are performed. Usually, the unstained quadrant should contain less than 3%. I need an explanation or a publication that explains why you can have high counts in the unstained quadrant.
Maybe you can give us more info. What type of cells or bacteria? which stains? fixed cells? intra or extracellular stains? are cells resistant to fluorescent probe?
Thanks Misagh and Dominic,
I microencapsulated Bifidobacterium longum in a lipid matrice using water in oil in water emulsion and then freeze dried. Now, the important thing is to find out how much of the cells remained viable at 4 degrees after few weeks. Basically, I had to break open the microparticles using dichloromethane, centrifuged , suspended in PBS, centrifuged again and re suspended in PBS before staining using a Live/Dead Baclight kit. This kit contains SYT09 (which stains all cells) and propidium iodide which stains only cells with compromised membrane. Both stains were used to differentiate live, dead and damaged cells. The attachment shows a four quadrant representation of the bacterial populations. Hope this helps.
Thanks for the info. This is interesting. B longum is microaerophilic anaerobe and I'm not sure how much of it survives all the processing, especially DCM. Are you making liposomes? Did you try triton X-100 to break up the lipids?
I see that Q2 is full of damaged cells, not sure if they are viable if you try to grow them and quantify those bacteria. Q1 is barely viable, Q3 is most unstained and I don't know why. Maybe you should fix your cells in 4% PFA after you extract them and then do live dead stain. I do kind of the same work as you, but I fix my bacteria before staining.
hope this helps.
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