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In the field of neuroscience, studying the functions of cerebrovascular and microvascular endothelial cells is crucial to understanding the normal physiological processes of the brain and the mechanisms of disease development. Primary brain microvascular endothelial cell extraction technology has been widely used, mainly by obtaining these cells from animal models, to deeply explore the structure, function and interactions of endothelial cells. This technology provides an important reference for studying the pathogenesis of related diseases and developing targeted therapeutic drugs, and promotes scientific progress in the field of cerebrovascular diseases and neurodegenerative diseases.

Welso carefully put together an experimental protocol for the separation and extraction of brain microvascular endothelial cells (BMECs) by using a low-speed centrifuge. The article aims to provide laboratory personnel with a concise and easy-to-understand technical guide, presenting in detail the key steps and precautions of the experiment, and helping scientific researchers quickly master the key points of the operation. We hope that through this sharing, we can provide practical guidance and support for research in related fields and accelerate the progress of cerebrovascular and neuroscience research.

Introduction to BMECs

Primary brain microvascular endothelial cells (BMECs) are an important type of cells isolated, cultured and expanded from brain tissue. They are mainly distributed in the walls of brain microvessels and constitute the core component of the blood-brain barrier. BMECs play a vital role in regulating the exchange of substances across the blood-brain barrier, maintaining the stability of the brain's internal environment, and defending against the invasion of harmful substances from the outside world. They are an important cell model for studying cerebrovascular function and disease mechanisms.

Experimental materials

Experimental animals: SD rats (4-6 weeks old, male)

Instruments and consumables: large surgical scissors, forceps, 50 mL centrifuge tubes, 6-well plates

Reagents and solutions: 75% ethanol, saline, collagenase, 25% BSA, poly-lysine

Culture medium: DMEM/F12 basal culture medium, DMEM/F12 complete culture medium

Laboratory instruments

Ultra-clean bench: for sterile operation

Low-speed centrifuge: for cell separation

CO2 incubator: provides a suitable environment for cell culture

Experimental procedures

Handling of experimental animals

●After anesthetizing the rats, soak them in 75% ethanol for about 1 minute for surface disinfection.

Obtaining the brain

● Cut the skin on the head, quickly and completely remove the brain, and place it in pre-cooled DMEM/F12 basal culture medium.

Cleaning tissue

● In a clean bench, remove the outer blood clots, meninges, and outer blood vessels of the brain (operate in a dish).

● Remove the white matter (loose structure) and keep the gray matter (shell-shaped left and right brain parts).

Washing

●Repeatedly wash the brain tissue using 4°C pre-cooled culture medium to remove impurities.

Cutting and preliminary processing

●Cut the brain tissue into small pieces of about 1 mm³ and transfer to a sterile 50 mL centrifuge tube.

●Further mince the tissue and gently pipette to disperse the tissue pieces.

Centrifugation

● Centrifuge at 900 rpm for 4 minutes at room temperature and discard the supernatant. Add 5 mL of serum-free DMEM/F12 medium and mix thoroughly with a pipette.

● Add 10 mL of medium and 100 μL of collagenase II and digest at 37°C for 30 to 45 minutes.

Prepare 6-well plate

●At the same time, use 1 mL of poly-lysine to coat the 6-well plate and incubate at 37°C.

Post-digestion treatment

●After digestion, add medium to neutralize, centrifuge at 900 rpm for 10 minutes at room temperature, and discard the supernatant.

●Add 15 mL of 25% BSA solution and centrifuge at 2000 rpm for 20 minutes at room temperature.

Extract target cells

● After centrifugation, stratification is visible: the dark red precipitate in the lower layer is the brain microvascular cells.

● Gently rinse the poly-L-lysine in the 6-well plate with saline and repeat the washing twice.

Cell resuspension and culture

● Remove the lower brain microvessel sediment, add physiological saline and mix well, then centrifuge at 900 rpm for 5 minutes at room temperature.

● Resuspend the cells with fresh DMEM/F12 complete medium, transfer to a coated 6-well plate, and culture in an incubator.

Through this experimental procedure, we successfully extracted and separated rat brain microvascular endothelial cells, providing a solid experimental basis for further research. Throughout the process, the low-speed centrifuge is a key tool that helps us efficiently separate cells and purify the target cell population. In order to ensure the accuracy and safety of the experiment, Welso has compiled some operating points of the low-speed centrifuge for reference by operators.

Equipment inspection and placement

● Ensure that the centrifuge is placed stably on a solid table to avoid instability caused by vibration or tilt.

● Check the equipment before use, and focus on checking the rotor and seals to ensure that there is no damage or looseness.

Operation protection

● Wear protective glasses and laboratory gloves during operation to prevent liquid splashing and causing damage to the skin and eyes.

Speed and load

● Pay attention to the maximum speed and maximum load of the equipment, and do not exceed the load range of the centrifuge.

● Select the appropriate centrifugation speed and time according to the experimental requirements. The speed and time should be adjusted according to the sample density, separation purpose and volume to avoid affecting the separation effect due to improper parameter settings.

Sample preparation and loading

● Before the sample enters the centrifuge, ensure that the centrifuge tube, lid, bucket and adapter are accurately balanced on the balance, the weight tolerance is generally not more than 1g, the precision requirement is not more than 0.1g, and it meets the requirements of the equipment manual.

● The materials filled in the centrifuge tube must have similar density to avoid vibration caused by uneven load distribution.

● The sample capacity must not exceed the maximum capacity limit of the centrifuge tube or rotor to prevent the equipment from being unbalanced or damaged.

●The sample should be placed symmetrically in the rotor to ensure that the centrifugal force is evenly distributed.

Operational Specifications

●It is forbidden to open or close the lid when the centrifuge is running to avoid accidental injuries.

●After centrifugation, make sure that the rotor stops completely before opening the lid. When taking out the sample, be careful to avoid contact with the high-temperature sample tube or splashing liquid.

Cleaning and Maintenance

●After use, clean the equipment and rotor in time to keep the equipment clean and avoid contamination or corrosion.

●Regularly check the operating status of the centrifuge to ensure long-term stability and reliability of the equipment.

●By following the above precautions, you can ensure the safety of experimental operations and extend the life of the equipment.

Centrifuges are essential in many cell separation and extraction experiments. Strictly following the centrifuge operating specifications can not only ensure the safety of the experiment, but also extend the service life of the equipment and ensure the reliability of the experimental results. Welso is a professional centrifuge supplier. You can learn more about the detailed features and technical parameters of low-speed centrifuges through our product page. We also welcome you to contact us at any time to get the most suitable quotation .