Application note written by RWD Life Sciences. Download.
Brain tissue contains complex and fragile neuronal networks. Conventional methods yield low sample viability with abundant fragments. A gentle enzymatic dissociation protocol, combined with debris removal, can yield high-quality single-cell suspensions suitable for various downstream applications. Particularly when paired with magnetic bead purification, it allows simple and rapid isolation of target cells.
Research background and significance:
The nervous system is the most complex and essential system in animal organisms. Its development involves processes such as neural induction, pattern formation, cell fate determination and specialization, establishment and migration of neural cell polarity, and the formation of neural circuits. Ultimately, these processes lead to the creation of a highly complex and functional neural regulatory network. To date, key questions related to nervous system development remain a focus of research.
Neonatal neural tissue dissociation
Materials and Methods
Reagents and equipment:
Neonatal Brain Tissue Enzymatic Dissociation Kit, Single Cell Suspension Dissociator.
Sample pretreatment
After stripping the neonatal brain tissue, remove blood capillaries gently from brain tissue.
Preparation of single-cell suspension
- Weigh the neonatal brain tissue and transfer it to the tissue processing tube incubated with Enzymatic reagent.
- Mount the tissue processing tube into the bushing of the single cell suspension dissociator, and install the heater.
- Run the program.
- Filter the cell suspension sample with the wetted cell strainer, and collect the cell suspension.
- (Optional) To remove red blood cells: Resuspend the cells resuspended with red blood cell lysis buffe, then incubate on ice.
Results
The cell viability of single-cell suspension cells from neonatal mouse brain tissue obtained was over 90%, with high cell yield and uniform results.
The dissociation of single cell suspension from neonatal mouse brain can be accomplished with ease using the the Single cell Suspension Dissociator in combination with the Neonatal Brain Enzymatic Digestion Kit.
Research background and significance:
Neurodegenerative diseases are chronic and irreversible neurological disorders caused by the progressive degeneration or death of neuronal cells in the peripheral or central nervous system. The main population affected by this disease is the middle-aged and elderly population. Currently, neurodegenerative diseases that have been discovered include Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). Aging is the main risk factor for neurodegenerative diseases. With the deepening of aging, neurodegenerative diseases seriously endanger the health of the elderly and cause serious socio-economic burden. Therefore, it is of great significance to find safe and effective treatment methods.
Separation of microglia from neural tissue dissociation:
Materials and Methods
Reagents and equipment
Adult Brain Tissue Enzymatic Dissociation Kit, CD11b+(Microglia) cell separation Kit.
Sample pretreatment
After stripping the adult brain tissue, hippocampus tissue, cortex tissue or spinal cord tissue, remove blood capillaries gently from brain tissue.
Preparation of single-cell suspension.
Weigh the adult and transfer it to the tissue processing tube incubated with Enzymatic reagent. Mount the tissue processing tube into the bushing of the single cell suspension dissociator, and install the heater. Run the program. Filter the cell suspension sample with the wetted cell strainer, and collect the cell suspension.
Debris removal using built-in reagents
(Optional) To remove red blood cells:
Resuspend the cells resuspended with red blood cell lysis buffe, then incubate on ice.
Separation of microglia
Take cell suspension. Add anti-mouse CD11b biotin antibody. Mix well and incubate at 2 ~ 8 °C.Wash cells with buffer, and discard the supernatant. Resuspend cells with buffer.Add Streptavidin MicroBeads.Mix well and incubate at 2 ~ 8°C. Wash cells with buffer, and discard the supernatant. Collect the CD11b + microglia.
Results
The cell viability of single-cell suspension cells from adult mouse brain tissue obtained was over 90%, with high cell yield and uniform results. And pure microglial cells with a purity greater than 95% can be separated from the single-cell suspension of the adult brain.
The dissociation and seperation of single cell suspension from adult mouse brain can be accomplished with ease using the the Single cell Suspension Dissociator in combination with the adult Brain Enzymatic Digestion Kit and CD11b+(Microglia) cell separation Kit.
