Most cell cycle assays amount to counting cells and/or measuring DNA content and deconvolving the resulting histogram into G1, S, G2+M, or G1, S, G2, and M.  Considerable effort has been put to correlating DNA content with RNA, protein, or specific epitopes to determine or discover additional compartments.  Here, we'll focus on flow cytometric analysis of DNA and specific epitopes.  In recent efforts, using DNA peak to mark anaphase/cytokinetic populations in a 6 parameter analysis (DNA-area, DNA-peak, Light Scatter, cyclin A2, cyclin B1, and phospho-S10-histone H3), we identify 15 compartments with more than 4 additional compartments representing apoptotic cells.

Rational approaches to fixation and optimized staining protocols were worked out years ago.  We have examined many experimental variations on the basic protocols without uncovering any major improvements that result in higher quality data.  We are working currently on a “washless” staining assay, relying on a single high dilution to minimize background staining and relying on acoustic focusing to render the sample analyzable over a short period.  The results are striking. The S/N is equivalent to a fully washed sample; the minimized handling results in better cell recovery, and improved recovery results in better definition of cell cycle compartments with low cell numbers.  Additionally, the “washless” assay provides significant labor savings.  For research use only.  Not for use in diagnostic procedures.

Learning Objectives:

• overview of fixation & staining for intracellular epitopes
• underlying logic and purpose of cell cycle analysis
• in depth, analytical protocol for multiparameter analysis backbone