This study focuses on the use of transgenic mouse mesenchymal stem cells engineered to express GFP and neurotrophic factors. The methodology includes immunofluorescence techniques to assess cell proliferation, indicating their potential for neurotherapeutic applications.
Begin with transgenic mouse mesenchymal stem cells—multipotent cells derived from bone marrow.
The cells are engineered to express GFP and therapeutic neurotrophic factors that support neuronal growth and survival.
Wash the cells with buffer and fix them to preserve cellular integrity.
Wash again with buffer, then add a solution to permeabilize the cell and nuclear membranes and block non-specific binding sites.
Introduce primary antibodies targeting a cell proliferation marker protein expressed exclusively in the nucleus of dividing cells.
Wash with buffer to remove unbound antibodies.
Add fluorophore-conjugated secondary antibodies targeting the primary antibodies and a DNA-binding dye to counterstain the nuclei.
Wash with buffer to remove unbound reagents.
Load the plate into the high-content screening system and capture images to detect fluorescent signals.
Expression of the cell proliferation marker indicates active cell division, suggesting the suitability of the transgenic cells for neurotherapeutic applications.
To carry out a Ki-67 cell proliferation assay, use 0.1 molar phosphate buffer to rinse the cell cultures for one minute. After repeating the wash, use 4% Paraformaldehyde, or PFA, at room temperature for 20 minutes to fix the cultures.
Following fixation, remove the PFA and use PBS to rinse the wells three times for seven minutes each. After blocking the cells according to the text protocol, apply 100 microliters of primary antibody solution to each well. Cover the plate and incubate at 4 degrees Celsius overnight.
After washing the cells three times for seven minutes for each wash, apply secondary antibody, place the cells in the dark, and incubate at room temperature for 90 minutes. Following another three washes, cover the plate and store it at 4 degrees Celsius until imaging.
To perform automated imaging, load the immuno-labeled plate into the HCS system and allow the plate to equilibrate for 20 minutes. Open the HCS system image acquisition and analysis software. Choose the acquisition settings for the 10X objective using camera binning at 1 and gain setting of 2.
Use the auto-exposure function to find the Z plane in which the cells reside, and calculate the offset for each wavelength of interest. For the analysis demonstrated here, capture images for DAPI, GFP, and Cy3.
Choose the maximum intensity level at which the negative control wells show no signal for image acquisition. Use the same threshold settings for positive wells. Finally, capture images and save them to a database, before performing image analysis according to the text protocol.
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