Hi, Awesome video. How are you preparing the dilution tubes though? Are you diluting them serially, or spiking in the precise number of ug to each tube? I'm asking because i'm doing a titration for the first time for a large panel, so many tubes, and i'm trying to figure out the easiest way to set up the tubes. Thanks!
Hi Lazar, Generally, I would say no. Most people titrate antibodies as a single stain. Doublet discrimination is important for ensuring that we're not getting false positives. Imagine if two single positive cells are stuck together - without doublet discrimination we might think we have a double positive cell. Because there's only one antibody to titrate in one tube the doublets aren't really a concern. Viability is also important for accuracy of your experimental data, your antibody might be nonspecifically binding to dead cells. See this blog for further explanation: voices.uchicago.edu/ucflow/2020/11/19/how-to-identify-problems-with-panel-design-bad-data-part-2/. For titrating an antibody the dead cells usually don't interfere too much with determining the optimal concentration. But I guess if you're seeing a much higher frequency of positive cells than expected or it seems like the background is really high maybe it's worth it to include a viability dye. I've yet to run into that situation, but I wouldn't say it's impossible. In that case you'd have to then worry about compensation between the antibody and viability dye, which is just more effort than a single stained tube. The simplest experiments are ideal.
Hi Claudia - Good catch, we'll figure out a solution on accessing the link on the slide. It's on the CAT Facility Blog: voices.uchicago.edu/ucflow/2020/01/16/how-many-cells-should-i-stain-the-impact-of-cell-concentration-and-antibody-concentration/
