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The nuclei shown in blue were stained with Hoechst The neurospheres were richly endowed with NPCs, which coexpress markers Nestin and Tuj The nuclei shown in blue was stained with Hoechst These neurospheres can be maintained for several weeks by culturing them in ultra-low attachment plates. A Phase-contrast image of neurons showing extensive sprouting. Transfer the 12 mm of sterile glass coverslips in the 24 well plates. This has given rise to the need for a more believable 3D model called neurospheres11, Neurospheres present a novel platform to neuroscientists, with a closer resemblance to the real, in vivo brain Neurospheres are non-adherent 3D clusters of cells that are rich in neural stem cells NSCs , neural progenitor cells NPCs , neurons, and astrocytes. Perform imaging of the fixed cells under a microscope at 10x and 40x magnification. Though primary neuron cultures i. Nuclei were stained with Hoechst blue. Remove all meninges from the hemispheres and midbrain by holding the brainstem. NOTE: The trypan blue dye exclusion method is based on the principle that live cells due to their intact membranes will exclude trypan blue dye and will hence show a clear cytoplasm, compared to a non-viable cell that will easily take up trypan blue and appear blue in color Dilute the number of cells obtained to plate 1. Culture the neurospheres obtained from the high-density plated neurons in the same maintenance medium by transferring them to the ultra-low attachment plates. Carefully remove the intact hemispheres resembling mushroom caps that contain the hippocampus and cortex. NOTE: Rats can also be euthanized by an overdose of pentobarbital or overdose of ketamine with xylazine or diazepam. NOTE: The anti-Mouse or anti-Rabbit secondary antibodies are selected depending on the host species of the primary antibody added. Wash the cells again with PBS once or twice. Examine the cells for adherence under the microscope 4 h after plating. A Cell morphology of the high-density plated neurons. Transfer the heads in the sterile dishes using sterile, blunt-ended dressing forceps. Comparatively more cell adherence was observed in the high-density plated neurons.

Life Science Solutions Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from EE16 Sprague Dawley Rat Embryo Gaurav Das1,2, Varsha Gupta1, Juhee Khan1, Deepshikha Mukherjee1, Surajit Ghosh1,2 1Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 2Academy of Scientific and Innovative Research AcSIR Abstract Primary neuron culture is an essential technique in the field micro mtt strategy neuroscience.

Here, the neurons have developed an elaborate synaptic network consisting of dendritic branches. Finally, to observe whether these neurospheres expanded over time, after 1 week of high-density culture, at which point the small neurospheres started to form, a few were transferred in ultra-low attachment plates and their growth was monitored every micro mtt strategy days for up to 15 days.

The most exciting aspect of primary neuron culture is that it creates an opportunity to observe and manipulate living neurons3. Transfer the coverslips in distilled water using a pair of forceps and swirl it gently to get rid of the acid completely.

Removal of brain and dissection of the cortex with hippocampus Before starting, fill 90 mm sterile Petri dishes with cold, sterile HBSS. They are an excellent source for the isolation of neural stem cells and neural progenitor cells, which can be used to study differentiation into various neuronal and non-neuronal lineages.

This includes a pair of blunt-end scissors, forceps, fine forceps, two fine scissors, and one artery forceps for the entire procedure.

Decapitate the head with sterile scissors. It should be kept in mind that the secondary antibodies must be conjugated to fluorescence derivatives suitable for fluorescence microscopy purposes.

Allow the digested tissues to settle down and aspirate the dissociation medium. Add 2. Moreover, it is less complex compared to neural tissue, which makes it an ideal candidate for studying the function and transport of various neuronal proteins. Primary cultures have allowed neuroscientists to explore the molecular mechanisms behind neural development, analyze various neural signaling pathways, and develop a more coherent understanding of synapsis.

B Cell morphology of low-density plated neurons. Plating medium: plating medium consists of the following: minimum essential medium MEM Eagle's with Earle's Balanced Micro mtt strategy Solution BSS; Combine the components in the respective ratios and perform the procedure inside a hood under sterile conditions.

Though a number of methods have reported cultures micro mtt strategy primary neurons mostly from the hippocampal origin5,6,7a unified protocol with a chemically defined medium that enables long-term culture of neurons is still needed.

For generating neurons and neurospheres, use a timed-pregnant Sprague Dawley rat and mark the day with vaginal plug detection as E0. Incubate the cells with 0. Add 4. Next, the percentage of astrocytes in the high and low density seeded cultures was assessed. These neurons can be further maintained for up to 30 days micro mtt strategy changing the maintenance medium every 3 days with the development of more intricate read article networks.

This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. NOTE: Always use freshly prepared plating medium to avoid degradation of any component. Seal the margins of the coverslip with dibutylphthalate polystyrene xylene DPX.

A-D Spontaneously generated neurospheres after 7 days in micro mtt strategy from the high-density plated neurons.

Aspirate the medium and add 10 mL of dissociation and plating medium consecutively to the digested tissues. However, glial cells pose a lot of problems due to their overgrowth, which sometimes override the neuronal growth Hence, considering the problems above, a simpler and more cost-effective primary neural culture protocol is required, which can be used by both neurobiologists and neurochemists for investigations. Preparation of coverslips Take a 12 mm diameter round glass coverslip and soak it in 1 M hydrochloric acid HCl for 4 h. Ensure that all components are freshly prepared. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. Characterize the neurons and the neurospheres by immunostaining them with important markers. Preparation of poly-D-lysine coated plates for neuron culture Take two 24 well plates: one for high density plating and another for low density plating. Take the embryonic sacs carefully on the Petri plate with cold HBSS solution. Some methods have even suggested co-culturing of the primary neurons with glial cells, wherein the glial cells are used as a feeder layer9. Collect the hemispheres containing cortex and intact hippocampus in a 15 mL conical tube containing 10 mL of dissociation medium. Reagent and media preparation Poly-D-lysine PDL solution: prepare PDL solutions at concentrations of 0. However, the inability of these clonal cell lines to form strong synaptic connections and proper axons or dendrites have shifted scientific interest to primary neuron cultures1,2. To do so, neuroscientists initially resorted to various transformed nerve-based cell lines for investigations. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day Sprague Dawley rats. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience. Insert the slide containing the mixture into the cell counter and obtain the reading. Purity of the neuronal culture is shown by the absence of staining of non-neuronal markers for GFAP of astrocytes Figure 3D and O4 of oligodendrocytes Figure 3E. Determine the density of viable cells using the trypan blue dye exclusion method and count the number of cells in an automated cell counter. Open the sterile packets only inside the laminar hood. Prepare 0. NOTE: The culture must be performed between EE Anesthetize an EE16 pregnant rat with an intraperitoneal i. A Cell Adherence in High Density plated neurons. Similarly, in the low density plated neurons, a primary neuron culture that can be maintained for up to 30 days is obtained by changing the maintenance medium twice per week. Introduction The brain is an intricate circuitry of neuronal and non-neuronal cells. Add 10 mL of fresh dissociation medium to the tissue, and repeat step 6. Collect all the embryo brains in the same manner in the HBSS solution. NOTE: Do not use the same forceps and scissors that were just used for the skin, as this will contaminate the internal organs. Since this methodology is aimed primarily at culturing neurons, it was important to assess whether this method supports preferential growth of neurons over non-neuronal cells, especially astrocytes. Figure 1: Representation of cell adherence after 4 h of plating. NOTE: Use the dissociation medium in ice-cold form during dissociation but at room temperature RT for washing and other purposes. Recently, several developments in the fields of microscopy, genomics, and proteomics have generated new opportunities for neuroscientists to exploit neuron cultures4. For years, scientists have been trying to gain insight into this complex machinery. The astrocytic population was also investigated through GFAP staining, compared to neuronal population Tuj1 staining in low density seeded cells, for 7 continuous days. The next day, wash the cells with PBS once or twice and add appropriate secondary antibodies in PBS at concentrations at RT for 2 h. Culture these neurons grown at low density for 30 days by changing the maintenance medium 2x per week. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Again, variability within neurosphere cultures produced using the previously reported protocols presents a barrier to the formulation of a unified neurosphere culture protocol This manuscript presents a protocol in which it is possible to generate both 2D and 3D platforms by alternating cell plating densities from a mixed cortical and hippocampal culture. Before using the coverslips, dry them well in the laminar hood by keeping them on tissue paper. Representative Results In this protocol, a simple strategy has been elucidated in which variable cell plating densities from two different neural screening platforms are obtained. Due to the lack of suitable media and nutrients to support its growth, this population of astrocytes also slowly perished over time, whereas in the presence of optimal factors and media, the neurons rapidly took over the entire culture. A primary neuron culture is essentially a form of 2D culture and does not represent the plasticity, spatial integrity, or heterogeneity of the brain. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. Under the stereomicroscope, hold the head from the snout region with sterile, serrated forceps and remove the brain by cutting the skin and skull open. Take the embryos out of the embryonic sacs in fresh, cold HBSS. To plot the line graph representing the eventual increase in the volume of neurospheres for each timepoint , 50 neurospheres were studied, and their averages were used to derive the neurosphere volumes at each timepoint.