I want to detect the presence of bacteria and antigens from the same sample using two fluorescent stains with flow cytometry. How can I double stain the sample for that purpose?
Hi Mohan, Bacteria will stain with Ho33342 DNA stain if you incubate 10ug/ml at 37C for 1h e.g. http://www.icms.qmul.ac.uk/flowcytometry/uses/bacteria/labho.gif. This dye is excited by violet or UV lasers & emits at 461nm. So if you have an antibody against bacteria or mammalian cells fluorescently tagged that is compatible with Ho33342 e.g. FITC, PE, APC you could then do this at RT for 20 mins. if you are looking at mixed cultures (I just did this yesterday) you can still set the FSC & SSC on Log and still get bacteria & cells on the plot. hope this helps Gary
Dear Gary,Thanks a lot. I am going to use Alexa Fluor 555. In this case which one should I add first? Staining bacteria and antibody reaction or staining the bacteria after antibody reaction? I found that SYTO9 is also used by a lot of researchers. Do you have any idea about SYTO 9?
Hi Mohan, yes you should be able to use SYTO9 to but I do not know the loading conditions for this, but you would need a Yellow-Green laser source for your AF-555 excitation. So I would load the SYTO9/Ho33342 into the bacteria first at 37C then label with the antibody next at RT. Hopefully there should be little spectral overlap with SYTO9 (488nm Ex) and AF-555 (561nm Ex). Regards Gary
Dear Gary, Thanks for the answer. I am going to use 488nm laser for this experiment and I am very new to flow cytometry. I checked the Alexa and SYTO 9 using the spectrum viewer by life technologies and it seemed OK. My main purpose is to determine the bacteria with antigens which can specifically interact with antibodies and to observe.
Dear Mohan, I see, but AF-555 is not excited by 488 nm, you will need to change the fluorophore to AF647 as the option AF488 falls into the same channel as SYTO9. If you only have a 488nm laser line you would need to change the antibody to PErCP, PE-Cy5.5 or PE-Cy7; if you have a red laser the easy option is to use AF647. Regards Gary
Dear Gary, Thanks for the answer. But still I am not very clear about the point. This is the image I am talking and the blue represents SYTO9 and Green is Alexa. As I understand, the laser 488 can excite both of them (the straight line is the laser). But if I use 633nm laser, it is out of this range. That is why I told I am going to use 488nm laser. Can you clarify this problem for me.
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