The overall goal of this procedure is to utilize neurosphere based culturing methods for primary neural stem and progenitor cells to study primary cilium biogenesis and trafficking. This method can help answer key questions in the primary cilia field such as cilia biogenesis and trafficking of cilia molecules to the primary cilia. In primary neural stem cells, neural progenitors and neurons.
The main advantage of this technique is that we can use the primary cell criteria of the neural stem cells, neural progenitors and neurons to analyze biogenesis and ciliary pathways in the context of the primary cilium. Generally, individuals new to this method will struggle because culturing neural stem cells is technically challenging for beginners. We'll show a demonstration of this method is critical, as dissection of the lateral ventricle and steps such as culturing neurospheres and quantification of primary cilia are easily learned by observing the critical steps.
Begin this procedure with a subventricular zone of the lateral ventrical dissected from an adult mouse using the whole mount dissection method. Place the tissue containing the SVZ into a 1.5 milliliter tube. Add 500 microliters of 0.05%Trypsin-EDTA in PBS and incubate 15 minutes at 37 degrees Celsius in a water bath.
Following the incubation, add 500 microliters of stopping medium and gently pipette 20 to 30 times using a 1 milliliter tip. Avoid forming air bubbles during pipetting. After this, spin down the cells at 500 times G for eight minutes.
Discard the supernatant then add 1 milliliter of PBS and resuspend the cells by gently pipetting five times using a 1 milliliter tip. Centrifuge at 500 times G for eight minutes. Then aspirate the medium using a 1 milliliter tip and add 1 milliliter of basal medium.
After counting the cells, plate the cells from one SVZ into a 10 centimeter dish with 10 milliliters of NSC medium and culture at 37 degrees Celsius with 5%carbon dioxide. After five to seven days, neurospheres can be observed. To analyze cells in attached neurospheres by immunostaining, transfer 1 milliliter of medium containing multiple neurospheres to a 1.5 milliliter tube and spin down at 500 times G for eight minutes.
After removing the medium, fix the spheres with 4%paraformaldehyde for 15 minutes. After removing paraformaldehyde incubate the spheres with 30%sucrose, overnight. And then centrifuge again.
Wash with PBS. Then, after centrifuging as before, remove and discard the supernatant. Then, fit a pipette with a cut 1 milliliter tip and pipette 500 microliters of OCT solution onto the neurospheres.
Transfer the OCT solution containing the neurospheres into a disposable plastic freezing mold. Then, freeze the mold on dry ice for at least 15 minutes. Cut sections with a cryostat.
To visualize the stem cells in the neurosphere perform immunostaining against the stem cell marker Nestin. To visualize the primary cilia in a neurosphere, perform immunostaining against ARL13B. Begin this experiment one to two days after seeding adherent dissociated neural stem cells onto Poly-L-Lysine and Laminin coated cover slips.
Change the medium in the experimental wells to starvation medium, and to fresh NSC medium in the control wells. Incubate for a further 24 hours. The next day, fix the cells with 4%paraformaldehyde in PBS for 15 minutes at room temperature.
After fixation, wash with PBS twice for five minutes each, at room temperature. And then perform immunoflourescence using standard protocols. Mount the cover slips with mounting solution.
Then, dry the slide glass overnight at room temperature in the dark. The next day, acquire images on a compound microscope at the necessary magnification. The microscope camera and objectives should be controlled using the accompanying software.
Take sufficient z-sections at 0.5 to 0.8 micron intervals. For the quantitative analysis of ciliary localization in adherent cells, select three to eight consecutive fields with confluent cells by viewing the DAPI channel. Acquire stacks of images from each field.
Quantify the number of primary cilia using ImageJ. Open the Plugins Analyze dialog box and select Cell Counter tool to count the cells with GPCR positive cilia. Use similar image intensity and contrast parameters for all images from the same experiment for counting and exporting purposes.
24 hours after culturing dissociated cells in NSC medium or starvation medium cells were fixed and immunostained against GPR161 shown in green, ARL13B which appears red, and DNA shown as blue. GPR161 positive cilia are observed in control cells on the left, and starved cells on the right. As seen in these enlarged views.
The white arrows and arrowheads indicate GPR161 positive and negative cilia, respectively. This image shows quantification of ARL13B positive ciliated cells. The quantification is performed from two fields of view each from two technical replicates of a single experiment.
The percentage of ARL13B positive ciliated cells increases upon starvation. This image shows quantification of GPR161 localizing in ARL13B positive cilia in control and starved cells. The percentage of GPR161 localizing in ARL13B positive cilia, also increases upon starvation.
Once mastered this technique can be done in less than two hours if it's performed properly. Following this procedure, another method of culturing primary tumor derived cells can be performed in order to answer additional questions like the role of cilia in tumor genesis. After its development, this technique paved the way for researchers in the field of primary cilia to explore ciliary biogenesis and trafficking in primary neurosphere based systems.
After watching this video, you should have a good understanding of how to analyze primary cilia in neural stem cells, neural progenitors and neurons that are derived from neurosphere based cultures.
