Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.

Department of Biomedical Engineering University of Arizona School of Plant Sciences BIO5 Institute Arizona Cancer Center Tucson Arizona USA

Laboratory of Growth Regulators Institute of Experimental Botany The Czech Academy of Sciences and Faculty of Science Palacký University Olomouc Czech Republic

School of Life Sciences Henan University Institute of Plant Stress Biology Kaifeng China

Umeå Plant Science Center Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences Umeå Sweden

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