In this section we provide several success ful protocols for sample preparation and cell sorting experiments. We detail the conditions in which samples are prepared for sorting and how the machines are setup and run for cell separation.
Cell Cycle analysis is one of the most applied protocols in flow cytometry, most commonly to measure frequencies of cells that are either in G0/G1, G2/M, or in the S-phase of the cell cycle. This can be done using a general DNA intercalating dye, such as Propidium Iodide (PI), to quantify the amount of DNA present in each cell. Cells in G0/G1 will typically have half of the DNA signal than cells in G2/M, and those in S-phase will have an intermediate signal between that of G0/G1 and G2/M cells.
Here we describe a few protocols to prepare several cell types for cell cycle analysis based on DNA content.
Non-Adherent Cells: Cell Cycle Analysis of Non-Adherent Cells by DNA Content using PI [.pdf]
Jurkat Cells: Cell Cycle Analysis of Jurkat Cells by DNA Content using PI [.pdf]
Recently developed through a research collaboration, this protocol allows the isolation of pure and viable fractions of each cell/nuclei type before and after pollen mitosis of Arabidopsislines. (ref: doi:10.1186/1746-4811-8-44)
Pollen: Purification of Microspores from young flower buds [.pdf]
Male germ unit: Purification of sperm cells, vegetative nuclei and sperm nuclei [.pdf]