Furthermore, an alternative method analyze cell proliferation was developed based on fluorescence decay tracking. Most recently, automated cell counters were developed to facilitate and accelerate the cell counting process. Formerly, it was performed using Neubauer chamber, which is a tremendously laborious and variable task. The most classical approach to generate growth curves is based on counting proliferating cells at different time points. Later on, CFSE was used to analyze the interference of drugs in cell lines proliferation and their doubling time. CFSE has become a powerful tool in the Immunology field. When stimulated, lymphocytes proliferate and each daughter cell receives half the CFSE content from the mother cell upon cytokinesis. CFSE was employed as a cell tracker in 1994 to identify proliferating lymphocytes after a stimulus.
It binds to free amine groups through the succinimidyl ester group and generates covalent dye-protein conjugates. Once the molecule is inside the cell, the acetate group is cleaved by cellular esterases and the resulting green fluorescent carboxyfluorescein molecule is no longer membrane permeable, unable to leave the cell. ĬFSE is a cell-permeant non-fluorescent pro-dye. In addition, cytoplasmic proliferation dyes, such as carboxyfluorescein diacetate succinimidyl ester (CFSE) and the cell trace violet (ThermoFisher Scientific, Cambridge, MA) have been employed to track proliferating cells.
Proteins associated with the cell cycle, such as Ki-67, phosphorylated-histone H3 and proliferating cell nuclear antigen (PCNA) are also used as cell proliferation reporters. Incorporation of nucleoside-analogues is used to identify cells in the S phase of the cell cycle, such as tritiated thymidine ( 3H-TdR) and 5-Bromo-2′-deoxyuridine (BrdU). The characterization of in vitro cell proliferation by plotting a cell growth curve may be assessed by different approaches. The most traditional assay to characterize in vitro cell proliferation is the cell growth curve. Currently, questions posed in several biomedical sciences fields may be addressed using the outstanding platform of in vitro proliferating cells. The development of in vitro cell culture technique provided the establishment of a variety of cell lines from different organisms, developmental stages and pathologic conditions. Moreover, the fluorescence-based method displays lower variation among different measurements of the same time point, which increases the reliability on the determination of lag, log and stationary phase transitions.
Therefore, this method is not biased due to either cell loss during harvesting or to the presence of cellular debris and cell clumps. The fluorescence-based method is not dependent on the determination of the total number of cells, but rather is approached by assessing the fluorescence of a sample of single cells from a cell population at different time points after plating. Here, we compare this traditional method to the fluorescence-based method, which is based on the CFSE fluorescence decay over time. Traditionally, growth curves for adherent cells are obtained by seeding the cells in multiple-well plates and counting the total number of cells at different time points. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions.