Experiment Overview
| Repository ID: | FR-FCM-ZZJL | Experiment name: | Quantitation of Cell-Cell Fusion Using the Multicolor FCM Assay II. Cell Wall Staining Optimization. Mating Pairs Subpopulation Identification. | MIFlowCyt score: | 92.38% |
| Primary researcher: | Valentina Salzman | PI/manager: | Pablo Aguilar | Uploaded by: | Valentina Salzman |
| Experiment dates: | 2014-08-07 - 2014-08-07 | Dataset uploaded: | Mar 2015 | Last updated: | May 2015 |
| Keywords: | [fertilization] [mating] [bi-molecular complementation] [Saccharomyces cerevisiae] | Manuscripts: |
|
||
| Organizations: |
Institut Pasteur de Montevideo, Montevideo, (Uruguay)
|
||||
| Purpose: | 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). | ||||
| Comments: | None | ||||
| Funding: | Not disclosed | ||||
| Quality control: | Each mating sample was analyzed by triplicate, unstained and compensation controls are included in the experiment. | ||||
Experiment variables
| Sample Type | |
|---|---|
| · wt control samples | 1_07_Aug_14.fcs · 20_07_Aug_14.fcs · 2_07_Aug_14.fcs · 36_07_Aug_14.fcs · 8_07_Aug_14.fcs |
| · prm1 control samples | 10_07_Aug_14.fcs · 11_07_Aug_14.fcs · 22_07_Aug_14.fcs · 3_07_Aug_14.fcs · 4_07_Aug_14.fcs |
| · wt X wt mating samples | 25_07_Aug_14.fcs · 26_07_Aug_14.fcs · 27_07_Aug_14.fcs |
| · prm1 X prm1 mating samples | 28_07_Aug_14.fcs · 29_07_Aug_14.fcs · 30_07_Aug_14.fcs |
| · ConA-Tet labeling optimization samples | 33_07_Aug_14.fcs · 38_07_Aug_14.fcs · 40_07_Aug_14.fcs |
| · ConA-647 labeling optimization samples | 34_07_Aug_14.fcs · 41_07_Aug_14.fcs · 43_07_Aug_14.fcs · 44_07_Aug_14.fcs |
| · ste2/3 control samples | 12_07_Aug_14.fcs · 13_07_Aug_14.fcs · 23_07_Aug_14.fcs · 6_07_Aug_14.fcs · 7_07_Aug_14.fcs |
| · MATaste2 X MATalpha ste3 mating samples | 31_07_Aug_14.fcs · 32_07_Aug_14.fcs |
| · sonicated samples | 12S_07_Aug_14.fcs · 13S_07_Aug_14.fcs · 20S_07_Aug_14.fcs · 23S_07_Aug_14.fcs · 31S_07_Aug_14.fcs · 32S_07_Aug_14.fcs · 6S_07_Aug_14.fcs · 7S_07_Aug_14.fcs |
