Quantitation of Cell-Cell Fusion Using the Multicolor FCM Assay II. Cell Wall Staining Optimization. Mating Pairs Subpopulation Identification.

The purpose number 1 of the experiment is to quantify cell-cell fusion by flow cytometric analysis of a wt X wt and a prm1∆ X prm1∆ yeast mating. 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. The purpose number 2 of the experiment is to identify the appropriate concentration range for each ConA-fluorophore conjugate. Haploid cells are stained and incubated at standard mating conditions. The purpose number 3 is to control whether the mating pairs subpopulation is being properly gated, a cell fusion assay between two sterile mutants MATa ste2∆ and MATalpha ste3∆ is performed under standard conditions. Conclusion: Cell fusion efficiencies of a wt X wt mating and a prm1∆ X prm1∆ mating were quantified (1). For our working system ConA-647 optimal concentration is 20 µg/ml and 250 µg/ml ConA-Tet (2). Mating pairs subpopulation is properly gated (3). Each mating sample was analyzed by triplicate, unstained and compensation controls are included in the experiment.