1. Differential Fluorescent Cell Labelling
2. Cell-cell Fusion
3. Fluorescence-activated Cell Sorting (FACS) to Enrich Fused Cells
4. Immunofluorescent Staining and Imaging of Cell-cell Fusions
NOTE: Fused cells can be imaged live or after fixation and further fluorescent staining (or both), depending on the experiments and measurements required.
Appropriately labelled cells are visible during flow cytometry by fluorescence signal higher than unlabeled control cells (Figure 2A). Gates are set for sorting of double positive cells, enriching this population directly into imaging dishes for further microscopic analyses. Fused cells are detectable as distinct double fluorescently positive cells and constitute about ~1% of the population.
Fusion induces major rearrangement of cellular architecture through mixing of two cells into one (Figure 2B). Heterokaryons are identified on the microscope as cells containing both fluorescent dye signals mixed inside a single cell (without intervening plasma membranes). Additionally, two nuclei may be visible in fused cells by brightfield/differential interference contrast or fluorescence imaging. Note that triploid or other more highly polyploid states are possible in addition to diploid fusions however, and so dye signal of two colors should be used to confirm the identity of fused cells.
Cell structure and function are further investigated through the merging of cells containing meGFP and mScarlet-I tagged proteins. Fusion results in a doubling of the centrosome number inside heterokaryons resulting from the fusion of two cells. Thus, if cells with fluorescently labelled centrosomes are fused, at least four pericentriolar material foci are observed when the centrosome pericentriolar component NEDD1 is fluorescently tagged (NEDD1-mRuby3; Figure 2C). Fusion of cells with endogenously fluorescently tagged rootletin (rootletin-meGFP and rootletin-mScarlet-I) allows the cell of origin of each centrosome to be identified in a heterokaryon. Rootletin in centrosomal roots has limited diffusional turnover24, and is therefore present as distinctly colored fibers in heterokaryons imaged with fluorescence microscopy (Figure 2D).
Figure 1: Cell-cell fusion and fluorescence imaging experimental workflow. Schematic of the four-stage experimental workflow. (1) Two cell populations are differentially labelled, with dyes and fluorescent fusion proteins. Cyan represents staining with violet cell dye and magenta represents staining with far red cell dye. Green represents meGFP tagging and red represents mScarlet-I tagging. (2) Cells are fused through incubation with polyethylene glycol. (3) Fused cells are enriched by flow cytometry, sorting the double fluorescently positive cells (far red and violet). (4) Fused cells are imaged by fluorescence microscopy to understand how cellular structure and function are altered (imaging the green and red channels). Please click here to view a larger version of this figure.
Figure 2: Representative flow cytometry enrichment and fluorescent imaging of fused cells. (A) Representative gating strategy used in flow cytometry sorting of fused cells. Fused cells that are doubly fluorescent are indicated by the black square. (B) Representative confocal fluorescence microscopy of fused cells, double labelled with violet and far red dyes. Shown are examples of successfully fused cells, which are either tetraploid or hexaploid (top and bottom panels respectively). Scale bar = 10 µm. (C) Representative live cell Airyscan confocal imaging of centrosomes in a single fused cell containing endogenously labelled centrosomal roots (rootletin-meGFP) and centrosomal pericentriolar material (NEDD1-mRuby3). Scale bar = 1 µm. (D) Cells expressing endogenously tagged rootletin-meGFP were fused with cells expressing endogenously tagged rootletin-mScarlet-I. Cells were fixed and stained and imaged by structured illumination microscopy. Shown is a maximum-intensity z-projection of centrosomes in one fused cell. Scale bar = 1 µm. Panel D has been modified from Mahen24 with permission. Please click here to view a larger version of this figure.
15 ml tube | Sarstedt | 62554502 | |
37% formaldehyde solution | Sigma-Aldrich | F8875 | |
880 Laser Scanning Confocal Airyscan Microscope | Carl Zeiss | ||
8-well imaging dishes | Ibidi | 80826 | |
Anti-GFP alpaca GFP booster nanobody | Chromotek | gba-488 | |
BD Influx Cell Sorter | BD Biosciences | ||
Bovine serum albumin | Sigma-Aldrich | A7906 | |
Cell Filters (70um) | Biofil | CSS010070 | |
CellTrace Far Red | ThermoFisher Scientific | C34572 | |
CellTrace Violet | ThermoFisher Scientific | C34571 | |
Dulbecco's Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate | ThermoFisher Scientific | 31966021 | |
Fetal Bovine Serum | Sigma-Aldrich | 10270-106 | |
FluoTag-X2 anti-mScarlet-I alpaca nanobody | NanoTag Biotechnologies | N1302-At565 | |
L15 CO2 independent imaging medium | Sigma-Aldrich | 21083027 | |
Penicillin/streptomycin | Sigma-Aldrich | 15140122 | |
Phenol red free DMEM, high glucose | ThermoFisher Scientific | 21063029 | |
Phosphate buffered saline (1 x PBS) | 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2HPO4, dH2O up to 1L | ||
Polyethylene Glycol Hybri-Max 1450 | Sigma-Aldrich | P7181 | |
Polypropylene tubes | BD Falcon | 352063 | |
Triton X-100 | Fisher BioReagents | BP151 | nonionic surfactant |
Trypsin | Sigma-Aldrich | T4049 | |
Tween 20 | Fisher BioReagents | BP337 | nonionic detergent |
Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.
Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.
Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.