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DAPI and Hoechst 33342 (there are different Hoechst dyes, 33342 is one of the most commonly used) have very similar spectral characteristics. The only point is that DAPI is much better excited at 405 nm than Hoechst. Some microscopes and flow cytometers nowadays have 405 nm lasers or LEDs instead of UV sources, so this can become relevant. The main ...


3

I don't believe that the Isotype Control Antibody makes the antibody bind more specifically but rather ensures that your signal is specific to your antibody. The Isotype Control Antibodies tend to bind to the non-binding domain of the antibody, typically the constant (Fc) domain. By measuring the concentration of the control antibody, you can determine what ...


2

I realize this is an older topic, but I'd like to clarify some misconceptions: 1) DAPI is usually referred to as a semi membrane-permanent dye because its penetration through viable cell membranes is concentration dependant. At lower concentrations it is mostly excluded from viable cells, while at higher concentrations it stains live and dead cells ...


2

The Hoechst 33342 dye is similar to DAPI in that both are UV-excited, minor groove-binding, and emit signals proportional to total DNA content. Both are maximally excited around 355 nm and emit around 460 nm. A UV light source is required, which may harm the cell. However, this is only a risk with Hoechst, as DAPI requires the cells to be fixed and/or ...


2

While bobthejoe/leonardo's answers are correct, you also need to be aware of the quality and limitations of isotype controls. You need to pick your source very carefully, as extensive testing (at my former company) has shown that some isotypes stick non-specifically under certain conditions or with certain samples. For example, we found that one common ...


1

Sounds like you should look into Bioconductor's flow suite. I actually really like doing flow analysis in R, but it takes some practice and getting used to. The biggest problem you will run into (in my opinion) is gating. It's not easy to setup a GUI point and click gate. Rather, you'll be more forced and inclined to set limits with algorithms or hard ...


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The answer I have come to is the difference between FCS2.0 and FCS3.0 files. The binning pattern is different, and as such FCS2.0 files arbitrarily lowered my flow cytometry values compared to what I saw on the FACS DiVa software. In the end, exporting with FCS3.0 was the way to go. If your software supports this, I'd highly recommend doing so!


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I have this issue too with data from a Partec PAS III. I don't consider it a problem, as it's really just the x-axis which is one order of magnitude higher. At the end, all values coming from the machine are arbitrary values, so as long as the change is systematic, there's no problem.



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