Hello,
I am currently setting up my compensation matrix in flow cytometry and to do this I am using compensation beads (versacomp antibody capture beads) to get negative and positive population per antibody that I'm using.
I have a primary antibody that is biotinylated and I used streptavidin APC secondary antibody for detection. I tried staining the positive beads by incubating it with the biotinylated primary antibody (20mins, wash) and then adding the streptavidin APC secondary antibody (20mins, wash). However, I did not detect any positive population at all.
Is there anyone that can explain to me why this is or how i can get a positive population using the beads.
Thank you in advance.
So... compensation beads are actually antibody coated. The one I use is coated with antibodies that recognize every possible rodent antibody that is commonly used: such as anti-mouse, anti-rat, anti-hamester,and anti-rabbit.
You don't need the primary. All you are doing is detecting signal. If your secondary is a mouse antibody, find a comp bead that is anti-mouse then just stain with the secondary. That should work.
Dear April Baral,
Streptavidin is not an antibody. See the link below.
https://en.wikipedia.org/wiki/Streptavidin
Then, it might be helpful to check the species of your primary antibody. For example, if it is mouse anti-human antibody, then the species is mouse. If it is rat anti-mouse antibody, then the species is rat, and so forth. You can check whether the species of your primary antibody matches the species supported by your beads.
By the way, there is an easier tip. In compensation using beads, you do not need to use the same antibody used for your sample. Instead, you can use any other antibody conjugated with APC given the species matches. For example, if you use mouse anti-human CD4-biotin and streptavidin-APC for your sample, you can use mouse anti-human CD8-APC for staining the compensation beads. I suppose this method could be easier if you have an appropriate antibody conjugated with APC.
Thank you Shen-An Hwang and Tetsuo Tsukamoto
So my primary antibody is a Murine Monoclonal Anti-Human antibody and should be supported by the beads I am using.
Just a follow up question Tetsuo Tsukamoto , if I use another antibody like you said i.e CD8-APC, would this affect my compensation set up if the MFI is different since I would be detecting different antigens? Or this would not affect it since APC signal (fluorescence) would give similar median from both APC-conjugated antibodies?
Regardless of whether you are using the same or different antibody-APC for compensation, the staining of compensation beads should be bright enough to be comparable to the staining of cells. If your beads are stained too dim, then that would not be effective as a compensation control. Of course, the staining should not be too bright either.
The average staining level of cells would be affected by both the target molecule density on cell membrane (x) as well as the antibody concentration. On the other hand, the average staining level of beads would be affected by the density of anti-mouse Ig antibody coating the beads (y) as well as the antibody concentration. I guess y is likely to be higher than x in most cases.
This also means that the difference in targeted antigen (regardless of whether its CD3, CD4, CD8, CD19, ...) would not affect compensation. Because fluorochromes are usually conjugated to antibody at the ratio of 1:1, if you use the same concentration of f.g. anti-CD8-APC or anti-CD4-APC or whatever, either should stain beads with nearly identical staining levels.
To summarize,
(1) Determine the optimal Ab/Streptavidin concentrations for your cells by a single stain and check the APC MFI of positive cells.
(2) Titrate anti-CD8 APC (or whatever) for the use with comp beads so that the MFI would be comparable to the above.
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