The presented protocol describing neurite outgrowth assay that is highly relevant to human biology and neurotoxicity assessment of a small molecule compounds. High translational potential and physiological relevance of human neural progenitor cells as the primary human cell model offer a considerable advantage in neurite outgrowth-related drug discovery screening and neurotoxicity assessment. Utilizing the presented protocol, human induced pluripotent stem cell and human embryonic stem cell-derived neurons can also be used as to alternative cell sources for the neurite outgrowth assay and neurotoxicity assessment.
Begin by collecting the media with floating neurospheres and transferring it to a 50 milliliter conical tube. Centrifuge the tube at 300 to 400 times g for three minutes. Then carefully aspirate the supernatant.
and resuspend the spheres in 500 microliters of defrosted cell dissociation reagent. Incubate the spheres at 37 degrees Celsius for five to 15 minutes. Then add five to 10 milliliters of pre-warmed culture media and centrifuge the tube at 300 to 400 times g for five minutes to sediment the neurospheres.
Aspirate the supernatant and add two milliliters of cultural media pipetting up and down with a 1000 microliter pipette until the neurospheres form a single cell suspension. After counting the cells, add two to 3 million cells to a T25 flask in 10 milliliters of culture media. Replace half the culture media every three days.
Use commercially available cryopreservation medium to freeze the cells. Transfer the neurospheres to a 50 milliliter tube and allow the spheres to settle at the bottom. Use a 200 or 1000 microliter pipette tip to transfer the big neurospheres to a new 50 milliliter tube before passaging.
Centrifuge the remaining neurospheres at 300 to 400 times g for three minutes and carefully remove the supernatant. Resuspend up to a thousand spheres in one milliliter of cryopreservation reagent and transfer it to a cryo tube. Store the tube overnight at minus 80 degrees Celsius, then move it to liquid nitrogen for long-term storage.
To measure neuronal outgrowth, open the image in Fiji and select Analyze, Tools and ROI Manager. Right-click on the fifth icon in the tool bar Straight and switch to Freehand Line. Optionally, double-click on the same icon to change the line width to 10.
Then trace the longest neurite beginning near the cell body and extending to the tip. Press Control and T then F to add the measurement to the ROI Manager and highlight the measured neuron. Select all numbers in the ROI.
Click on Measure, then copy and paste the measurements into a spreadsheet. To measure fluorescence intensity of labeled cells, click on the fourth icon in the toolbar Freehand Selections and draw the shape of the cell. Click Analyze and Set Measurements.
Then make sure that Area, Mean gray value, Min max gray value and Integrated density are selected. Click Analyze and measure. Select the region next to the cell as background.
And then click analyze and measure. Select all measure data and copy, paste it into a spreadsheet. To coat the plate add 30 microliters of poly-L-lysine into each well of a 384-well plate and incubate the plate at room temperature for one hour.
Wash it twice with PBS and let it dry for approximately 30 minutes. Add 30 microliters of laminine to each well, then incubate the plate at 37 degrees Celsius for two hours. Repeat the washes with PBS and proceed with plating the cells.
Add 20, 000 single cell neurospheres in 25 microliters of differentiation media to each well of the 384-well plate and incubate the plate at 37 degrees Celsius for five days. Hight precision pipetting skills are required for plating the exact number of neurospheres that's all segregated into single cells. Therefore, differentiating the neurospheres into neurons before plating is recommended to achieve more reproducible results.
After the incubation, add five microliters of test compound to each well at six times the desired concentration. And incubate the cells for another 24 hours. To perform the cell viability essay, add 30 microliters of luminescent reagent to each well.
And leave the plate on a shaker for two minutes. Centrifuge the plate at 300 to 400 times g for 30 seconds, then incubate the plate at room temperature for 10 minutes, protecting it from light. After the incubation, measure the luminescence on a microplate reader.
The human neural progenitor cell-derived neurons were used to examine the effect of HDAC inhibitors as small molecule epigenic compounds for the extension of neurites and subsequent neurogenic ability of small molecule compounds. The neurotoxicity of the HDAC inhibitors was also assessed after differentiating the neural progenitor cells in 384-well plates, showing the potential to scale the protocol for a higher number of compounds. In a different study, measurement of the neuronal cell fluorescence intensity was used to quantify the abundance of histone H4 lysine five acetylation as a histone marker after treating the neurons with epigenetic modifier compounds.
Additionally, this protocol has been used to visualize a pre-synaptic protein, synaptophysin, to check for possible synaptogenic effects of small molecule compounds. Using the presented method, neurites number, intensity, length, and width, together with synaptic characteristics, including pre and post-synaptic protein modifications could be reliably measured.
