A Guide to Choosing Fluorescent Protein Combinations for Flow Cytometric Analysis Based on Spectral Overlap

About the Presenter

Andre Olsson, PhD
Research Associate
Lee Grimes Lab

Andre received his PhD in experimental hematology at the Faculty of Medicine, Lund University in Sweden. In graduate school he studied the function of the ETO (Eight Twenty One) homologues and found that they are involved in regulating hematopoietic progenitor maintenance and lineage differentiation. As a research associate at Lee Grimes lab, he has focused on understanding myeloid lineage decisions during homeostasis. Hematopoiesis is a great system for studying cells and the processes that instruct the cells how to function. Flow cytometry enables people to study cells at a single cell level, and thus is a very powerful tool to map and characterize cells going through differentiation.

Webinar Summary

Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. The advent of facile genome engineering technologies has made the generation of gene-expression or fusion-protein reporters more tractable. While there are a number of fluorescent proteins available, their choice as reporter constructs is made difficult by the lack of data on how sensitivity and other factors are affected when two or more fluorescent proteins are combined. We characterize the detection sensitivity, spectral overlap, and spillover spreading of 13 monomeric fluorescent proteins to determine their utility in multicolor panels.

Learning Objectives

• Describe how to consider fluorescent protein detection sensitivity for fusion-protein studies.
• Learn how spectral overlap and spillover spreading impacts which fluorescent proteins to combine in an experiment.
• Discuss how experimental validation is key to successful panel design.

Who Should Attend

Anyone interested in using fluorescent proteins in their in vivo or in vitro research.

CMLE Credit: .5