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It goes without saying that at some point in performing immunohistochemistry (IHC) experiments on formalin-fixed paraffin embedded (FFPE) tissue, you will come across an antibody that just does not seem to work. After carefully checking to make sure you properly titered your primary antibody, confirmed your secondary antibody does recognize the species in which your primary antibody was made, and tried digesting your tissue with multiple enzymes, you might give up ever trying to get this antibody to work!

Virtually all flow cytometers are equipped with a blue (488 nm) laser and configured to measure fluorescence in the conventionally known ‘FL3’ detector. Over the years, a number of fluorochromes have been introduced for use with this detector including the PE tandem dyes, PE-Cy5 and PE-Cy5.5, as well as the small fluorescent protein Peridinin chlorophyll (PerCP) and its tandem dyes, PerCP-Cy5.5 and PerCP-eFluor® 710. So many choices!

You took great care in the design of your multicolor staining panel, taking into consideration expression levels of your antigens of interest, potential compensation issues…..all those issues you have been trained to consider! Never-the-less, when you actually ran your sample, some of the compensation values were much higher than you expected and the data looked a bit messy – what’s up with that? Using fluorescent beads to calibrate your instrument and set ‘optimal’ PMT voltages does not always translate to ‘optimal’ instrument settings for the particular staining panel you are using. In our example below, the staining panel includes a PerCP-Cy5.5 conjugate (B220) and a PE-Cy7 conjugate (CD3). As can be seen in the figure below, there is some overlap in the emission spectra for these fluorochromes that will have to be dealt with through proper compensation.

What does ED50 mean?

We all think we know what the ED50 value of a recombinant protein is; in short, it is the concentration at which the protein exhibits 50% of its maximum activity. Simple enough, right? It is our go-to piece of information on the data sheet when we consider a protein for purchase, but how many of us are truly familiar with what it means? Below are some guidelines for understanding the uses and limitations of ED50 values.

Apoptosis represents a highly regulated multibiochemical event. This pathway plays a key role in cellular development, like Hematopoiesis, and differs from necrosis in that apoptosis is controlled cell death.

If you design staining panels with upwards of 5 or 6 fluorescent parameters, you likely experience the love-hate relationship that many researchers have with tandem dyes. These frequently maligned fluorochromes do require a little more care and consideration than the seemingly indestructible small molecule fluorochromes, but with a little effort, that love-hate relationship can tilt toward the love side! As described in our blog article on "Tandem Dye Myths Debunked", tandem dyes do not ‘fall apart’, but conditions such as light exposure and bubbly buffers may oxidize the dye which leads to a loss of FRET efficiency and, hence, more fluorescence from the donor dye.

Have you ever wanted to use a flow cytometry tool that allowed you to:

• View a picture of multicolor spectral overlap for a specific cytometer laser/filter configuration?
• Develop an optimal laser and filter configuration for an existing multicolor experiment of known conjugated antibodies and dyes?
• Identify the ideal laser and filter setup to collect the brightest fluorescence signal and reduce compensation?

Tandem dyes have been used in flow cytometry for decades and have allowed for progressively larger numbers of markers to be investigated in a single experiment.  Myths about tandem dyes exist: we have all heard or said “the tandem fell apart” or there is lot-to-lot variability due to “inconsistency in the manufacturing process.”  There are also some additional considerations for using tandem dyes in multi-color flow cytometric experiments. Before addressing these myths and considerations, a little background about tandems.

We all use antibodies in western blotting, flow cytometry and IHC just to name a few applications. In each assay we use 1 uL or a 1:100 dilution, right? If that doesn’t work as we had anticipated, then we increase the amount. Hopefully that works. If it doesn’t work, then what do we do? Who has the time or extra samples to optimize the concentration?

At eBioscience, we are always reading papers to stay up on the latest trends and to know where our customers are directing the future of science.