Quantitation of Cell-Cell Fusion of wt Non Sonicated Samples Using the Multicolor FCM Assay II. Validation of Compensation Control

The purpose number 1 of the experiment is to quantify cell-cell fusion by flow cytometric analysis of a wt X wt. MATa and MATalpha strains are distinguished by staining each strain with ConcanavalinA- Alexa Fluor 647 (ConA-647) and with ConcanavalinA- Tetramethylrhodamine (ConA-Tet), respectively. Mating pairs are revealed as two-colored entities. Cytoplasmic mixing is measured with a GFP bi-molecular fluorescence complementation assay. Cell fusion efficiency is calculated as the percentage of fused mating pairs over the total number of pairs. Samples are sonicated before flow cytometry analysis. The purpose number 2 is to verify whether the used compensation controls are the proper ones for this cell fusion analysis. We include in the standard fusion assay experiment single-stained fusion samples obtained from wt x wt mating crosses of strains harboring the same GFP fragment where only one population is stained.
Conclusion:
Cell fusion efficiency of a wt X wt non sonicated mating was similar to sonicated samples (1). Haploid single-stained samples are suitable controls to set up compensation parameters, enabling the performance a complete analysis of a cross with only two strains (2). Mating was analyzed by triplicate, unstained and compensation controls are included in the same experiment.