2 – Reagents

Flow cytometrists use the Jablonski diagram to aid in understanding and explaining the kinetic events of fluorescence. Fluorescent compounds start at the ground state until they are excited by interacting with a photon of light. This photon excites the compound, promoting an electon to a higher energy state. Some of this energy is lost by emission of heat and other non-radiative processes, leading to the previous energy state. Finally, an electron falls back to the ground state while releasing a photon of light. This photon has a lower energy (higher wavelength) than the exciting photon of light. Here's how understanding…

The field of flow cytometry is moving beyond the use of isotype controls, with many suggesting they be left out of nearly all experiments. Yet, isotype controls were once considered the only negative controls you should ever use. They are still very often included by some labs, almost abandoned by others, and a subject of confusion for many beginners. What are they, why and when do I need them? Are they of any use at all, or just a waste of money? Most importantly, why do reviewers keep asking for them when they review papers containing flow data? Here is…

Sudoku puzzles seem to be all the rage. I see it in coffeehouses, at the airport, even in doctors offices. Everyone is trying to work out how to fit the numbers into the grids so that everything adds up properly. Designing polychromatic flow cytometry panels is much like the Sudoku puzzle. In this case, the grid is composed of the antigens on one side, and the cytometer detectors on the other. The goal is to fill in the grid correctly.   Instead of adding up to 45, like in Sudoku, the flow cytometrist is trying to optimize the ability to…

Pairing highly expressed antigens (like CD3) with dimmer fluorochromes, and the antigens of interest with the brightest fluorochromes, is a key part of panel design with few tools to help. With early generation instruments, this was relatively easy to determine, since fluorochrome choice was limited. With the advent of instruments capable of measuring more than 4 fluorochromes, there is a need to characterize the relative brightness of different fluorochromes under actual experimental conditions, rather than as free fluors. Bigos et al (2004) first reported this in an abstract and it was later simplified in Maecker et al (2004). This equation (Figure…

When we learn about fluorescence, the first thing we are told is that fluorophores emit photons that are higher wavelength than the photons that they absorb. What this specifically refers to is the stokes shift, which results from non-radiative energy transfer during the fluorescence process. When a photon is absorbed by a fluorophore molecule, some of the resultant energy is lost in molecular vibration and movement (among other things) so that the energy released after fluorescence is lower than the energy absorbed. Since wavelength is inversely proportional to energy, this lower output energy light is higher in wavelength than the…

I was at a meeting talking about the principles of panel design. At the end of my talk, I had an investigator approach me and ask why he was not making progress on his 15-color panel that he started developing. So, I asked how long he’d been working on it. A month. That was his response. This might shock some of you but a month is not very long when it comes to designing an accurate and effective antibody panel for a flow cytometry experiment. Multicolor panel design requires a delicate balance of biology and physics. Understanding the biology of…