Method for separating mouse brain tissue single cells

Abstract

The invention discloses a method for separating single cells of mouse brain tissues, which comprises the following steps: sterilizing killed mice with alcohol, obtaining complete mouse brain tissues in an ultra-clean workbench, cutting the brain tissues into pieces, digesting the brain tissues for 2-3 times by adopting 0.25% pancreatin, and filtering digestive juice to obtain cell suspension after the digestion is ended; centrifuging the cell suspension to obtain a cell precipitate, and resuspending the cell precipitate by using a Percoll solution with the volume concentration of 19-21%; adding a Percoll solution with the volume concentration of 36-38% into a separation tube to serve as a bottom layer, then adding the cell precipitation resuspension to serve as a middle layer, finally adding a NaCl solution with the mass volume concentration of 0.85%, and performing centrifugal washing to obtain single cell precipitation; according to the method, the cells are separated directly through Percoll gradient centrifugation, tedious operation and reagent dependence are avoided, the risk of cell injury is reduced, the operation time is shortened, the steps from brain taking to single-cell suspension obtaining are reduced, and the method is suitable for high-throughput experiment requirements.

Description

Technical Field

[0001] The invention relates to a method for separating single cells of mouse brain tissue, belonging to the technical field of single cell separation. Background Art

[0002] In neuroscience research, single-cell isolation technology of brain tissue is an important foundation for building in vitro cell culture models, analyzing the cellular mechanisms of complex diseases, implementing high-throughput drug screening, exploring gene function regulation mechanisms, and developing cell therapy strategies. Especially in the field of adult mammalian brain tissue research, single-cell resolution analysis has become a key technical means to reveal the fine structure of neural circuits, dynamic developmental processes, and cellular heterogeneity in pathological states.

[0003] At present, the isolation of single cells from adult mouse brain tissue mainly relies on enzymatic digestion combined with density gradient centrifugation. The traditional method has the following technical bottlenecks: 1) It relies on brain tissue perfusion to remove circulating cells, which has a long operation cycle, and improper control of cardiac perfusion pressure can easily cause mechanical damage to neuronal axons, resulting in reduced cell survival rate; 2) Red blood cell lysis requires multiple centrifugation and washing (usually ≥3 times), which not only increases the complexity of the operation, but also causes up to 30% loss of nucleated cells; 3) Some experimental methods for isolating single cells from mouse brain tissue do not remove the myelin sheath and directly perform density gradient separation. The purity of the obtained single cells is not enough, and there are many myelin impurities that affect subsequent experiments, especially during flow sorting, which is easy to clog the flow cytometer; 4) The existing Percoll density gradient separation system uses a three-stage concentration configuration of 30%, 37%, and 70% (total dosage is about 9mL / sample), which has high experimental costs. The number of cells obtained from the separation of an adult mouse brain tissue is about 300,000, and the cell yield is usually low, which makes it difficult to meet the technical requirements of high-throughput analysis such as single-cell sequencing. Summary of the invention

[0004] In response to the problems existing in the prior art, the present invention provides a method for isolating single cells from mouse brain tissue. The method can achieve the separation and preparation of single cells from adult mouse brain tissue with a high cell yield without the need for perfusion or red blood cell disruption. The method simplifies the separation process and has high compatibility. It is particularly suitable for applications in the fields of high-throughput single-cell omics research in the field of neuroscience, cellular mechanism analysis of complex disease models, and neural stem cell research.

[0005] The method for separating single cells from mouse brain tissue of the present invention is as follows: After killing the mice by cervical dislocation, the mouse carcasses were sprayed with alcohol for disinfection, and then the fur on the mouse heads was quickly removed in a clean bench, the skulls were peeled off, and the brain tissues were completely removed; the removed brain tissues were gently rinsed in 1× PBS buffer containing double antibodies and 1× PBS buffer without double antibodies in turn, and finally, the olfactory bulbs, brain stems, and part of the meninges and blood vessels were carefully peeled off in DMEM-F12 basal culture medium to separate and obtain brain tissue samples; 2. Cut the brain tissue into pieces and digest it twice in 0.25% (w / v) trypsin (trypsin is a proteolytic enzyme that can cut the connecting proteins between cells and disperse the cells in the tissue into single cells). The digestion was terminated with DMEM-F12 medium containing 10% FBS, and the digestion solution was filtered through a 40μm cell sieve to obtain a cell suspension; 3. Centrifuge the cell suspension to obtain a cell pellet, and resuspend the cell pellet with a 19-21% volume concentration Percoll solution; first add a 36-38% volume concentration Percoll solution as the bottom layer in a separation tube, then add the cell pellet resuspension as the middle layer, and finally add a 0.85% mass volume concentration NaCl solution, centrifuge at 900-1000g, 15-20°C for 25-35min, collect the bottom liquid, add a 0.85% mass volume concentration NaCl solution to wash, centrifuge, repeat 1-2 times, and obtain a single cell pellet; The 36-38% Percoll solution has a higher density and is located at the bottom of the separation tube; the cell pellet resuspension (including cells and impurities) is placed in the middle layer; the NaCl solution with a mass volume concentration of 0.85% is located at the top layer; after centrifugation, the demyelinated cells will settle to the 36-38% Percoll solution layer due to their higher density, while the myelin fragments have a lower density and will float on the top layer (0.85% NaCl solution and 19-21% Percoll liquid layer), thereby achieving accurate separation of demyelinated cells and myelin fragments.

[0006] Advantages and technical effects of the method of the present invention: 1. No need for complex pretreatment, simplifying the experimental process: omitting the perfusion and red blood cell lysis steps. The traditional method requires perfusion to remove blood and lyse red blood cells, while this method directly separates cells by Percoll gradient centrifugation, avoiding cumbersome operations and reagent dependence, reducing the risk of cell damage, shortening the operation time, and reducing the steps from brain extraction to obtaining single cell suspension, which is suitable for high-throughput experimental needs; 2. High cell yield and high survival rate: The layered centrifugation method using 19-21% and 36-38% Percoll solutions combined with physiological saline can accurately separate demyelinated cells (located in the 36-38% layer) and myelin fragments (located in the top layer). About 1.5 million cells can be obtained from one brain tissue, and the survival rate of cells stained with trypan blue is about 95.7%; Low-damage enzyme digestion: two gentle digestions with 0.25% trypsin combined with 40μm filter ensure cell integrity and reduce cell damage compared to mechanical grinding; 3. Wide compatibility and application potential: The single cells obtained by the method of the present invention can be used for flow cytometry and further used for the sorting of microglia (CD11b + CD45 - , yield 9.87%)); it can also be used for complex disease model research, such as survival detection of neurons transfected with Tdtomato after transplantation and separation of cells marked with specific markers. The method of the present invention is suitable for functional research of primary nerve cells; 4. Experimental cost and repeatability advantages The amount of Percoll reagent used is small, saving costs; the method is simple, easy to operate and has good reproducibility; While ensuring high cell activity and high yield, this method greatly simplifies the experimental steps, reduces technical barriers and costs, and provides an efficient and reliable solution for high-throughput single-cell omics research and disease mechanism analysis in the field of neuroscience, which has practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 Schematic diagram of the morphology of a single cell taken under an inverted microscope after the single cell pellet was resuspended (10X), wherein Figure A is a cell obtained by the method of Example 1, and Figure B is a cell obtained by the method of Comparative Example 1; Figure 2 Figure 1 shows the flow cytometry results of microglia. Figure A shows the flow cytometry results of FITC-H and PE-H detecting FITC (CD11b) and PE (CD45) bound to the surface of microglia. The area encircled by the oval is the microglia population CD11b. + CD45 low , Figure B shows CD11b + Results of regional microglia positivity rate; Figure 3 Schematic diagram of neurons with red fluorescence 48 hours after tdTomato plasmid transfection (10X), Figure A is a 10X schematic diagram of primary hippocampal neurons with red fluorescence taken under a fluorescence microscope 48 hours after plasmid transfection, Figure B is a primary hippocampal neuron photographed in bright field, and Figure C is a schematic diagram of primary hippocampal neurons after the bright field and red fluorescence are merged; Figure 4Figures A, B, and C are the negative control groups, and Figures C, D, and E are the tdTomato plasmid transfection groups. The circled cells in P1 area in Figures A and D are living cells, the circled cells in P2 area in Figures B and E are single cells, Figure C M3 is the negative control, and Figure F M3 is the rate of positive cells with red fluorescence in the tdTomato plasmid transfection group. DETAILED DESCRIPTION

[0008] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified; Example 1: The method for isolating single cells from mouse brain tissue is as follows After killing C57BL / 6J mice by cervical dislocation, spray the mouse carcasses with alcohol for disinfection, then quickly remove their fur in the clean bench, and carefully peel off the skull, because the outside of the mouse brain is covered with hard bones, to ensure that the brain tissue is completely removed. Next, the removed brain tissue is gently rinsed in 1× PBS buffer containing double antibodies and 1× PBS buffer without double antibodies. Finally, carefully remove the olfactory bulb, brain stem, and part of the meninges and blood vessels in DMEM-F12 basal culture medium to obtain brain tissue samples; 2. The brain tissue was minced and digested twice in 0.25% trypsin, and the digestion was terminated with DMEM-F12 medium containing 10% FBS. The digestion solution was filtered through a 40 μm cell sieve to obtain a cell suspension; 3. Prepare Percoll stock solution (mix 9 parts of commercially available Percoll separation solution with 1 part of 8.5% NaCl (Sigma) to achieve physiological osmotic pressure). Dilute the Percoll stock solution with 0.85% NaCl physiological solution to prepare 20% and 37% Percoll solutions by volume. 4. The cell suspension was centrifuged to obtain a cell pellet, and the cell pellet was resuspended with 3 mL of 20% Percoll solution. In a 10 mL centrifuge tube, 3 mL of 37% Percoll solution was first added as the bottom layer, and then the cell pellet resuspension was added as the middle layer. Finally, 2 mL of 0.85% NaCl solution was added. The tube was centrifuged at 900 g and 18 °C for 30 min. The myelin-containing liquid (0.85% NaCl solution and 20% Percoll liquid) was carefully aspirated with a 5 mL syringe, and the bottom 37% Percoll solution (containing demyelinated cells) was collected. 8 mL of 0.85% NaCl solution was added for washing, and the tube was centrifuged at 1200 rpm for 6 min. This was repeated once to obtain a single cell pellet. According to statistics, about 1.5 million single cells were obtained.

[0009] Comparative Example 1: 1. Steps 1 and 2 of this embodiment are the same as those of embodiment 1; 2. Prepare Percoll stock solution (mix 9 parts of Percoll separation solution with 1 part of 8.5% NaCl (sigma) to achieve physiological osmotic pressure). Dilute the Percoll stock solution with 0.85% NaCl physiological solution to prepare Percoll solutions with volume ratios of 30%, 37%, and 70%; 3. The cell suspension was centrifuged to obtain a cell precipitate, and the cells were transferred to 3 mL of 37% Percoll solution; 3 mL of 70% Percoll solution was first added to a 15 mL centrifuge tube, and then 37% Percoll solution containing cells was added, and finally 3 mL of 30% Percoll solution and 2 mL of 0.85% NaCl solution were added in sequence; centrifuged at 300 g and 18 ° C for 40 min. After centrifugation, the demyelinated cells were located at the boundary between the 37% and 70% Percoll gradients, and the myelin sheath was located at the top of the tube, that is, in NaCl. Demyelinated cells were collected from the 37% and 70% gradient interface and washed twice with 6 mL of 0.85% NaCl to remove the remaining Percoll; the number of cells obtained was about 300,000, which shows that the comparative method not only has a high reagent cost, but also has a lower cell quantity than the method of the present invention.

[0010] Example 2: The single cell pellet obtained in Example 1 was used for flow cytometry and further used to sort microglia 1. Resuspend the single cell pellet of Example 1 with 1 mL of flow cytometry wash buffer (1×PBS) to obtain a single cell suspension and count the cells. Figure 1 , after statistics, about 1.5 million single cells were obtained; 2. Divide the single cell suspension into four equal parts and add them into 1.5 mL conical EP tubes. Use wash buffer (1×PBS) to make up the insufficient liquid volume to 1 mL. Centrifuge at 2000 rpm / min for 4 min. 3. Discard the supernatant, add 1 mL of wash buffer (1×PBS) to each well to resuspend and wash the cells, and centrifuge at 2000 rpm / min for 4 minutes; 4. Discard the supernatant and perform extracellular staining. Set up negative control, single staining and double staining groups. Add 0.25μL of microglia antibody CD11b (FITC) and 0.25μL of CD45 (PE) and Facs medium (2% FBS in 1×PBS) to the 1.5mL EP tube; add 50μL of Facs medium (2% FBS in 1×PBS) to the negative control tube; add 0.25μL CD11b antibody to the CD11b single staining tube, and make up the rest of the volume with Facs medium to 50μL; add 0.25μL CD45 antibody to the CD45 single staining tube, and make up the rest of the volume with Facs medium to 50μL; add 0.25μL of CD11b antibody and CD45 antibody to the double staining tube, and make up the rest of the volume with Facs medium to 50μL. After resuspending the four tubes, incubate at 4℃ in the dark for 30min; 5. Add 1 mL of Wash buffer to each well to wash the unbound antibody, centrifuge at 2000 rpm / min for 4 min, discard the supernatant, add 1 mL of Wash buffer to each well to resuspend the cell pellet, centrifuge at 2000 rpm / min for 4 min, discard the supernatant, then resuspend with 100 μL of Wash buffer and place on ice for testing; 6. Flow cytometry was used to detect that under normal physiological conditions, CD11b and CD45 on the surface of microglia would show specific expression patterns. Anti-CD11b antibodies were labeled with FITC and anti-CD45 antibodies were labeled with PE. When these labeled antibodies combined with the corresponding antigens on the surface of microglia, they would generate fluorescence signals after laser excitation. FITC-H and PE-H parameters can accurately reflect the intensity of fluorescence signals, thereby identifying cells expressing CD11b and CD45. Since microglia express CD11b and CD45 expression levels are low (CD11b ＋ CD45 low ), based on the signal intensity of these two parameters, microglia can be accurately located among numerous cells; Results Figure 2 This method can successfully isolate microglia, which account for 9.87%. The isolated microglia are used in subsequent reverse transcription experiments to reverse transcribe the mRNA in the cells into cDNA, and then use quantitative PCR technology to accurately detect the expression level of specific genes, helping researchers understand the expression differences of genes in different tissues, different developmental stages or different disease states.

[0011] Example 3: Application of the method of the present invention in complex disease model research 1. Transplantation of primary neurons transfected with tdTomato plasmid into mouse brain Primary neurons isolated and cultured from mouse brains are grown in T25 flasks to a confluence of 70-80% and can be transfected. On the second day, prepare the transfection system: Mix1: 10μg tdTomato plasmid is added to opti-MEM, mixed, and the volume is 0.5mL; Mix2: 20μL lipofectamine 2000 is added to opti-MEM, mixed, and the final volume is 0.5mL. Incubate at room temperature for 5min, and leave it at room temperature for no more than 20min. Mix Mix1 and Mix2, mix gently, and incubate at room temperature for 20min. During the incubation period, the primary neurons were washed with 1× PBS, and 2-3 mL of opti MEM was added and placed in the incubator. 1 mL of the above-mentioned mixed system was added to the primary neurons. After culturing for 5 h, the old culture medium was removed and 4 mL of neuron-specific culture medium (48.5 mL Neuro Gro neuron basal culture medium + 1 mL B27 + 0.5 mL 50 mmol / L glutamine) was added. The cells were placed in an incubator at 37°C and 5% CO. 2 Continue to culture in an incubator under the same conditions; 48 hours after transfection, primary neurons can be observed under a fluorescence microscope with red fluorescence. Figure 3 As shown in the figure, the results show that the plasmid transfection was successful, and the neurons were marked with red fluorescence and can be used for brain injection; 0.25% trypsin digestion was used to collect the cell pellet and counted. The collected primary neurons were positioned in the CA3 area of ​​the hippocampus of the C57 mouse brain using a Reward brain positioning injection instrument. The coordinates were (M / L+2mm, D / V-2mm, A / P-1.67mm). A microinjector was used to inject 1 million cells into each mouse in each group, 500,000 cells in each left and right brain; 2. After 10 days, refer to steps 1-4 of Example 1 to obtain a single cell pellet; 3. Resuspend the single cell pellet in step 2 with flow cytometry wash buffer (1×PBS) to obtain a single cell suspension; 4. Detection by flow cytometry, first separate live cells and single cells by side scatter (SSC) and forward scatter (FSC). FSC-A can evaluate cell integrity. Normal intact cells have specific size and morphology. The volume of damaged cells will change, and the FSC-A value will also change. If the cell ruptures and the contents flow out, the volume decreases, and the FSC-A signal weakens, the health of the cell can be judged. Neurons have complex internal structures and many particles, and the side scatter light they produce is strong, and the SSC-A value is high. Therefore, live cells in single cell suspensions are selected with FSC-A as the horizontal coordinate and SSC-A as the vertical coordinate. The role of FSC-H: FSC-H is mainly used to determine whether the cell is a single cell or a cell cluster. In the process of separating mouse brain tissue cells, cell clustering may occur, affecting the accuracy of the analysis. The FSC-H signal can help identify cell clusters. The FSC-H signal of cell clusters will be higher than that of single cells. Therefore, single cells in single cell suspensions are selected with FSC-A as the horizontal coordinate and FSC-H as the vertical coordinate. Cells carrying the tdTomato plasmid will emit red fluorescence. In the graph with PE-A as the horizontal axis and count as the vertical axis, the cell population with fluorescent signals (i.e. PE-A has a value) is likely to be the target cell subpopulation. In the negative control (isolation of brain tissue from an adult mouse that has not been injected into the brain), no corresponding fluorescent signal peak will appear, which helps to determine whether the isolated cells contain the expected cell type. The results are shown in Figure 4 , exogenous neuronal cells survive in the mouse brain (the method of the present invention can be used to sort cells injected into the mouse brain and detect the survival rate of exogenous cells in the mouse brain). It provides a powerful experimental method for neuroscience research and has a wide range of applications in many fields such as neural development, neural disease mechanisms, drug development, and neural regeneration: fluorescently labeled single cells can be used to track the development of neurons in the mouse brain and help analyze the neural development mechanism; it can also provide a basis for exploring the pathogenesis of neural diseases and drug development by observing the changes in labeled single cells under disease conditions.

Claims

1. A method for isolating single cells from mouse brain tissue, characterized in that: After killing mice and sterilizing them with alcohol, obtain the complete mouse brain tissue in a clean bench, cut the brain tissue into pieces, and digest it with 0.25% trypsin for 2-3 times. After the digestion is terminated, filter the digestion solution to obtain a cell suspension; centrifuge the cell suspension to obtain a cell pellet, and resuspend the cell pellet with a Percoll solution with a volume concentration of 19-21%; First add a 36-38% volume concentration Percoll solution as the bottom layer in a separation tube, then add the cell pellet resuspension as the middle layer, and finally add a 0.85% mass volume concentration NaCl solution. Centrifuge at a centrifugal force of 900-1000g and 15-20°C for 25-35min, collect the bottom liquid, add a 0.85% mass volume concentration NaCl solution to wash, centrifuge, repeat 1-2 times to obtain a single cell pellet.

2. The method for isolating single cells from mouse brain tissue according to claim 1, characterized in that: Filter using a 40 μm cell sieve.

3. The method for isolating single cells from mouse brain tissue according to claim 1, characterized in that: After washing, centrifuge at 1200 rpm for 5-7 min.

Images