In mammals, autophagy is regulated by the mammalian target of rapamycin (mTOR) and is carried out by autophagy-related (ATG) proteins.
Turnover of macromolecules not only removes unwanted, malfunctioning or potentially harmful materials and structures it also provides for replenishing metabolic intermediates, and, in addition, some intermediate breakdown products supply also materials like antimicrobial peptides and antigenic fragments for presentation by innate immune cells. With this process, autophagy satisfies multiple requirements. Manner in which these data are extracted may be different for different software.As summarized in our preceding article and described in detail in excellent reviews, autophagy is a catabolic process responsible for turnover of macromolecules and organelles through lysosomal degradation. These data can be exported, tabulated andĪnalyzed in a suitable presentation package, for example, Excel or Prism. It is possible to analyze each parameter (FSC, SSC, red and green fluorescence.Red-to-green fluorescence intensity ratio (R/GFIR) of individual events Proportion of cells above selected threshold can be reported as fold For the control group, delimit a positive population – above the threshold –įrom 5 to 10 % and, with the same threshold setting, analyze the other.Threshold (R/GFIR-BT) along the population axis, so that the red-togreenįluorescence ratio is considered, which may be determined by a linearĮquation 𝑦 = 𝑎x + b, where the slope (a) can be set according to the In the linear scale dot-plot set an R/GFIRbased Percentage of events above a defined threshold Setting up the machine, collect at least 5.000 gated events. Linear scale plot, the events are positioned as represented in the figures along the dot This voltage will allow sufficient space along the axes for the most red-stained cells toīe held in the graph and not over-spill the end. Then be fine-tuned so that in the log scale plot, intensities will be between 10 2 and 10 3. SSC photo multiplier tube voltages so that the majority of dots in the first two-parameter dot-plot are containedĪdjust the red and green fluorescence detectors voltage up or down. Using the appropriate setting panel, adjust FSC and Introduce the sample and set the machine to “run”. Plot a two-parameter dot-plot with linear scale to detect red fluorescence (y-axis) and A small percentage of cells may remain unstained.
AUTOPHAGY FLATICON SOFTWARE
Some software do not permit concurrently selecting two scales (logarithmic and linear) for the same parameter. Select the correct bandpass filters, according to the figure 1 of the manuscript, to detect red fluorescence (y-axis) and green fluorescence (x-axis) Plot a two-parameter dot-plot with logarithmic scale to gate out unstained cells. Plot a two-parameter dot-plot of Forward Scatter (FSC) vs Side Scatter (SSC) and continue with standard procedures to gate out debris and doublets Incubate the cells for 15 min at room temperature in the dark and proceed with the acquisition on the flow cytometer.Resuspend the cells by gently pipetting - all samples should be prepared in a single-cell suspension – and transfer to the tube/dish for flow cytometry acquisition Add the medium with AO in the proportion 2:1 (AO medium:enzymatic solution) to a final concentration of 1 ug/mL AO.Harvest the cells by enzymatic release and dissociate cell aggregates.Prepare the AO staining solution by diluting the stock solution in pre-warmed culture medium (AO medium) in a concentration of 1.5 ug/mL.Grow cells on the desired cell culture plate and induce the cells according to your specific protocol.Perform the staining procedures in subdued light Prepare a 500 ug/mL stock solution of Acridine orange in ddH 2O, store at 4 oC and protect from direct light.This information is to serve as a guide and may need to be optimized for particular cell type or flow cytometer equipment. This is the standard protocol used for the preparation of adherent culture cells used for flow cytometric analysis in this paper.
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