Background Regular methods for quantifying IncuCyte ZOOM? assays involve measurements that evaluate how quickly the initially-vacant region turns into re-colonised with cells as a function of period. and is unaffected by EGF relatively. Results Our strategy for calculating and from an IncuCyte Move? assay provides even more fine detail about cellular-level actions than regular strategies for analysing these assays. In particular, our strategy can become utilized to evaluate the stability of cell cell and migration expansion and, as we show, enable us to evaluate how the addition of development elements impacts these procedures separately. as a function of period [13C15]. The relatives wound denseness can be a percentage of the filled region of the initially-scratched region to the total region of the scrape [12]. To AG-1024 demonstrate this typical approach we present a series of images from an IncuCyte ZOOM? assay with PC-3 cells [16] in Fig. ?Fig.1.1. PC-3 cells are a prostate cancer cell line with high metastatic potential [16, 17]. The experimental image in Fig. ?Fig.11(?(a)a) shows the initial scratch, and the subsequent re-colonisation of the initially-vacant area is shown in Fig. ?Fig.11(?(bb)C(d). The data in Fig. ?Fig.11(?(e)e) demonstrates the temporal variation in the relative wound density, which is automatically calculated by the IncuCyte ZOOM? system [12]. While this kind of standard approach for quantifying IncuCyte ZOOM? assays can provide useful information about how quickly a particular cell population is able to re-colonise the initially-vacant area, it does not distinguish between the relative roles of various cellular functions. The collective spreading of a population of cells is driven by both cell motility and cell proliferation [1C4, 18]. However, traditional data extracted from IncuCyte ZOOM? assays does not give us any indication of the relative roles of cell motility and cell proliferation. This additional information could be important in terms of understanding Rabbit Polyclonal to LY6E how a particular growth factor or a potential drug treatment affects collective spreading since it is possible that the addition of a growth factor or drug treatment could affect: (i) cell motility alone, (ii) cell proliferation alone, or (iii) both cell motility and cell proliferation, simultaneously. Fig. 1 Images from the control IncuCyte ZOOM? assay with PC-3 cells showing, AG-1024 (a) the initial position of the scratch, and the subsequent collective cell spreading after 12, 24 and 36 h in (b)C(d), respectively. Scale bar corresponds to 300 … In this methodology article we describe an alternative method for interpreting IncuCyte ZOOM? assay data using a continuum mathematical AG-1024 model. Our approach allows us to quantify the rate of cell migration in terms of an undirected cell diffusivity, and for these cells. Under control conditions our method gives and increases monotonically with EGF concentration whereas we observe a nonmonotonic relationship between and EGF concentration, with a maximum proliferation rate when the assays are treated with 50 ng/mL EGF. Although the techniques described here have been used previously to calibrate mathematical models to experimental data from circular barrier assays [18, 21, 22], this is the first time that IncuCyte ZOOM? assay data has been used to calibrate these parameters, and the first time that this process has been used to quantify how estimates AG-1024 of and depend on the concentration of EGF in an IncuCyte ZOOM? assay. Methods IncuCyte ZOOM? Assay We perform a monolayer scratch assay using the IncuCyte ZOOM? live cell imaging system (Essen BioScience, MI USA). This system measures scratch closure in real time and automatically calculates the relative wound density within the initially-vacant area at each time point. The relative wound density is the ratio of the occupied area to the total area of the initial scratched region. All experiments are performed using the PC-3 prostate cancer cell line [16], which is obtained from the American Type Culture Collection (ATCC, Manassas, USA). Cells are routinely propagated in RPMI 1640 medium (Life Technologies, Australia) in 10 % foetal calf serum (Sigma-Aldrich, Australia), with 110 u/mL penicillin, 100 (ATCC). Cells are removed from the monolayer using TrypLE?(Life Technologies) in phosphate buffered saline, resuspended in medium and seeded at a density of 20,000 cells per well in 96-well ImageLock plates (Essen BioScience). After seeding, cells are grown overnight to form a spatially uniform monolayer. We use a WoundMaker?(Essen BioScience) to create uniform, reproducible scratches in all the wells of a 96-well plate. After creating the scratch, the medium is aspirated and the wells are washed twice with fresh medium.