Aggregate Discrimination of PI Labeled Cells
A single cell suspension is critical for precision and accuracy in the analysis of cell cycle data. Doublet discrimination is employed for submitted samples that have aggregates. This technique cannot compensate entirely for a poorly prepared single cell suspension. The current protocol for removing cell doublets maintains that two attached G1 cells passing through the laser beam will create fluorescent peak heights and pulse areas similar to a single G2 cell. An example of two methods used in this application is displayed in the data below. The PI signal is picked up on FS vs. PMT5 for DNA content and depicted as ungated. Standard separation of aggregates employs displaying the peak height vs. peak area (Peak vs. Integral). One advantage employed here is the ratiometric method of displaying the peak height divided by the peak area (Ratio) vs. peak area (Integral). Greater separation translates into greater discrimination and more confidence in reducing the contribution of clumped cells. In flow cytometry it is imperative that cellular clumps or debris greater than 100mm be removed by filtration or they will block the 100mm quartz flow cell. The results are a downed instrument unavailable to process other samples at the minimum or more costly in time and money is replacement of the flow cell. At the bottom of the page are some distributors of nylon mesh usually sold in sheets or bolts. Also Fisher Scientific distributes a Falcon 12X75mm tube designed for flow cytometry purposes (unsterile) with a 35mm cell strainer in the cap (Falcon # 2235/cat. # 08-771-23). Preparing viable single cell suspensions is important especially with adherent cells. If cell clumping or aggregates pose a problem with typical enzymatic detachment such as trypsin, analyzing bare nuclei or using a commercial reagent such as Accutase from Innovative Cell Technologies, Inc. which is a sterile formulation designed specifically to replace trypsin might be worth investigating as a solution. To learn more on the development of doublet discrimination see references below.
