![]() Provides quantitative analysis of axonal density and myelination (as proportion): the axonal marker SMI-31 (568nm) and myelin marker MOG (Z2, 488nm) were stained. To obtain the absolute number of double-positive (red and green) cells this proportion should be multiplied with the absolute number of red objects ( Count_RedObjects). Measurements exported are the counts of identified objects in all channels as well as the proportion of green objects contained within red objects ( Mean_RedObjects_Children_GreenObjects_Count). Subsequently, green objects contained within red objects are determined, signifying proliferating oligodendrocytes. In the red and blue channels nucleic objects are identified whereas the green channel gets a threshold applied, small features (Olig2) or myelin strings (AA3) are suppressed and remaining objects identified. The pipeline extracts red, green and blue channels from the input image and corrects for illumination. Pictures were taken with 20x magnification. ![]() Furthermore, the amount of proliferating oligodendrocytes (EdU+/Olig2+ or EdU+/AA3+) is identified. dapi-edu-aa3/ olig2.cpīoth pipelines quantify the absolute number of cells (as in dapi.cp), absolute number of proliferating cells (EdU Alexa Fluor 594), and absolute number of oligodendrocytes (Olig2 or AA3 stained for 488nm, respectively) as in olig2-aa3.cp. The only measurement exported is the count of nuclei. The blue channel of the input image is extracted, illumination correction applied, and nucleic objects detected. dapi.cpĬounts the number of stained nuclei (DAPI, EdU Alexa Fluor 594) in both culture systems for pictures in 20x magnification. Furthermore, for each input image an output image in PNG format is generated, visualising the extracted data with the purpose of easy manual verification of the data generation process. A trailing number in the file name (before the extension) is extracted as variable and the subfolder name is stored as – these are reflected in the output file ( DATA.csv). tif image files in the specified input folder and contained subfolders. Images were saved as TIFFs at 300dpi (1392x1040 pixels). Images were captured using an Olympus BX51 fluorescent microscope with standard filters for DAPI, GFP and C圓. To analyse cell proliferation, cells were pulsed with EdU and stained using the Click-iT EdU Alexa Fluor 594 Kit following the manufacturer's instructions. Bound antibodies were visualized using appropriate combinations of species/isotype specific fluorochrome-conjugated secondary antibodies (1:500, Invitrogen, either 488nm or 568nm), followed by mounting in a media containing DAPI. Primary antibodies used were the following: SMI-31 (1:1500, Abcam), Z2 (anti-MOG, 1:200, clone Z2), PLP/AA3 (1:100, hybridoma supernatant supplied by S. Cell cultures analysed are either dissociated myelinating cultures, neurosphere-derived astrocytes generated from striata of P1 Sprague-Dawley rats, or oligodendrocyte cultures purified from P1 rat cortex.Īfter a few days in culture, cells were fixed with 4% PFA and immunostained for different markers. These pipelines are used to characterise different glial cell cultures to quantify neurite density and myelination, differentiation of oligodendrocytes, proliferation and nuclei as well as cell densities (pixel intensity in various channels). Linington at the Institute of Infection, Immunity and Inflammation, University of Glasgow, UK. A collection of pipelines for the CellProfiler cell image analysis software, developed with the neuroimmunology research group of Prof C.
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