More Effective PRDX4 Methods Simplified

For example, despite fixative being added to the cells before applying the Cell Viability Indicator the vehicle control cells retained enough esterase activity to generate a strong fluorescent signal, indicative of the presence of a relatively large population of viable cells without compromised membranes at the time the cells were fixed, in contrast to the dose-dependent loss of signal obtained from the test samples that had been treated with cytotoxic compounds Nocodazole or Staurosporine (Fig. ?3B3B). Since the iCell Neurons, when fixed, tended to more slowly convert the Cell Viability Indicator they were either incubated longer or a 2x amount of the Cell Viability Indicator was applied to these cells. Fixed cells could also be stored for later analysis. Fluorescence Plate Reader Detection and Analysis Relative cell viability and neurite outgrowth was measured Duvelisib research buy using a Tecan Safire2 fluorescence plate reader (Tecan Group Ltd., Maennedorf, Switzerland) to detect the fluorescence intensity from each well, expressed in relative fluorescence units (RFU). Bottom-read fluorescence detection was performed using monochromator excitation/emission settings of 483/525 nm (12 nm bandwidths) for the green fluorescent Cell Viability Indicator and 554/567 nm (5 nm bandwidths) for the orange-red fluorescent Cell Membrane Stain. Cell-free controls were included and used for background subtraction. Unless stated otherwise, cell treatments were typically performed in triplicate, from which the mean �� SEM was plotted. Curve fitting Selleckchem Bleomycin was performed with GraphPad Prism (GraphPad Software, La Jolla, CA) using a nonlinear regression equation for variable slope sigmoidal dose-response to estimate 50% effective or inhibitory concentration (EC50 or IC50) values. Imaging The antibodies used for immunocytochemistry were obtained from Life Technologies. Cells were visualized using a Zeiss Axiovert 25 CFL inverted fluorescence microscope (Carl Zeiss, PRDX4 Oberkochen, Germany) and standard FITC or TRITC filter sets. Green and orange-red fluorescent images were captured using an integrated Pixera Penguin 600CL camera (Pixera, San Jose, CA) and processed using ImageJ software (NIH, http://imagej.nih.gov/ij) to create merged picture overlays. RESULTS AND DISCUSSION Neurite outgrowth is commonly monitored via immunocytochemistry followed by microscopic image acquisition and analysis, procedures which are both time-consuming and tedious (Fig. ?1A1A) [7, 8]. To generate a simpler method (Fig. ?1B1B) for rapidly approximating neurite outgrowth in addition to neuronal cell health, a select combination of fluorescent dyes and a background suppression reagent was evaluated as follows.