title: Assessment of GFP expression and viability using the tali image-based cytometer.
abstract: Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements(1). Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations(2). Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer(3). The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis(4-6) and cell viability analysis with propidium iodide (PI)(7-11). Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population. A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.
authors: Department of Molecular and Cell Biology, Life Technologies. Krissy.Remple@lifetech.com
authors: Krissy
authors: K
authors: Remple
authors: Laurel
authors: L
authors: Stone
mesh_terms: D000818:Animals; D001859:Bone Neoplasms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016466:CHO Cells; D045744:Cell Line, Tumor; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation* / Q000379:methods*; D012516:Osteosarcoma / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D013194:Staining and Labeling / Q000379:methods; D014161:Transduction, Genetic
chemical_list: D049452:Green Fluorescent Proteins