22 results about "Erythrocyte lysate" patented technology

The invention belongs to the technical field of regenerative medicine and biology, and particularly relates to a human glioma organoid isolated culture kit and a human glioma organoid culture method. The human glioma organoid isolated culture kit comprises a tissue activity protection solution, a P1 buffer solution, a human glioma organoid culture medium, an organoid cryopreservation solution and a red blood cell lysis buffer, and the culture method is carried out by adopting the human glioma organoid isolated culture kit. The human glioma organoid can be cultured by adopting the human glioma organoid isolated culture kit and the culture method, and the kit and the culture method have the advantages that the human glioma organoid grows quickly and can be cryopreserved and resuscitated.

The application discloses a kit for predicting the curative effect of immunotherapy on prostate cancer patients, which comprises reagent A, reagent B, reagent C, reagent D, reagent E and reagent F; the reagent A is a PBS buffer solution; the reagent B is a lymphocyte separation medium; the reagent C is a red blood cell lysis solution; the reagent D is a cell surface CD3 flow antibody; the reagent E is a cell surface CD19 flow antibody; and the reagent F is a 1% polyformaldehyde solution in terms of mass percentage concentration. The application further discloses an application method of the kit, which comprises the following steps: collecting venous blood of prostate cancer patients; extracting peripheral blood mononuclear cells (PBMC) in the venous blood of the prostate cancer patients; incubating the flow antibody; and performing flow cytometry analysis. The kit and the application thereof are simple and fast, non-invasive, repeatable, low in cost and short in detection time.

The present invention relates to the field of biomedical technologies and provides a method for efficiently capturing neutrophils from peripheral blood for single-cell sequencing by lysing red blood cells. The method includes the following steps: collecting human peripheral blood into an anticoagulant tube containing ethylenediaminetetraacetic acid; taking the ethylenediaminetetraacetic acid anticoagulated whole blood, adding a red blood cell lysate, lysing at room temperature, waiting until the turbid mixed blood solution becomes clear and transparent, waiting for 1 minute, then adding a FACS cell resuspension solution to terminate red blood cell lysis, and centrifuging; adding the FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding the FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding a phosphate buffer solution to slowly resuspend the cells, and centrifuging. By selecting a suitable anticoagulant, precisely controlling the red blood cell lysis conditions, and performing meticulous cell resuspension and centrifugation operations, the present invention can ensure high neutrophil activity and a low cell debris rate, provide a suitable single-cell suspension for single-cell RNA sequencing, and help analyze the characteristics and functions of various neutrophil subsets.

The invention discloses a method for extracting and separating cord blood and placenta stem cells of sika deer, and belongs to the technical field of biomedicine. The problems that an existing stem cell extraction and separation method is low in efficiency, poor in cell activity, low in purity and large in pollution risk are solved. The method comprises the following steps: collecting cord blood and placenta after delivery of sika deer; diluting the cord blood, performing density gradient centrifugation, collecting the albuginea layer enriched with the stem cells, adding an erythrocyte lysate to remove residual erythrocytes, and centrifuging and collecting the stem cells; the method comprises the following steps: cutting placenta tissues into pieces, putting the placenta tissues into trypsin-EDTA digestive juice containing collagenase, carrying out ultrasonic-assisted digestion, and centrifuging to obtain stem cells enriched at the bottom of a centrifugal tube; and finally, resuspending the collected stem cells in a culture medium containing fetal calf serum and growth factors, adjusting the cell concentration, and inoculating into a cell culture bottle for culture. According to the method, the extraction efficiency and quality of the sika deer cord blood and placenta stem cells can be remarkably improved, and the activity and safety of the cells are ensured.

The application discloses an enhanced red blood cell lysis solution, a red blood cell DNA extraction kit and a method. The application provides an enhanced red blood cell lysis solution, which can improve the yield of red blood cell DNA. The application also provides a red blood cell DNA extraction kit and discloses a method for separating or extracting DNA from red blood cells. In addition, the application also discloses applications of the kit and the method for separating or extracting DNA from red blood cells, and the applications include applications in detecting cancer or predicting the risk of suffering from cancer. The enhanced red blood cell lysis solution and the kit provided by the application have important scientific value and application prospect.

The invention provides a method for rapidly detecting a neutrophil extracellular trapping net. The method comprises the following steps: collecting peripheral blood; the method comprises the following steps: taking peripheral blood, adding different fluorescein-labeled anti-CD66b and CtiH3 antibodies and a dsDNA dye SYTOX Green, uniformly mixing, and incubating; adding a red blood cell lysis buffer, uniformly mixing, and incubating at room temperature; adding PBS, uniformly mixing, and centrifuging; the method comprises the following steps of: removing supernatant, adding PBS (Phosphate Buffer Solution) to resuspend cells, detecting by a flow cytometer, detecting CD66b positive cells as neutrophils by adopting an APC channel, detecting CtiH3 as CtiH3 positive neutrophils by adopting a PE channel in combination with a neutrophil population, detecting dsDNA as dsDNA positive neutrophils by adopting an FITC channel in combination with the neutrophil population, and calculating a detection result according to an NETs formation index calculation formula. The invention also provides a system for evaluating the NETs of the sample to be tested. According to the invention, the detection time of NETs detection is shortened, and the stability and diagnosis efficiency of NETs detection are improved.

The present invention discloses a reagent composition for identifying mouse fibroblast subpopulations and a method for using the same, relating to the field of cell technology. The reagent composition includes an antigen blocking solution, an allophycocyanin (APC)-labeled Ly6C antibody, a phycoerythrin (PE)-labeled CyclinD1 antibody, a fluorescein isothiocyanate (FITC)-labeled DCN antibody, a PBS phosphate buffer, a cell digestion solution, and an erythrocyte lysis solution. The present invention can identify and sort DCN-positive fibroblast subpopulations in the lung tissue of mice with acute lung injury, or identify and sort DCN-positive fibroblast subpopulations in the lung tissue of mice with acute lung injury that are undergoing tissue damage or repair, providing conditions for in-depth research on the role and mechanism of DCN-positive fibroblasts in ARDS, and exploring clinical treatment or prognostic targets.

The invention discloses a lymphatic endothelial cell cyst fluid extraction method for lymphatic deformity, and belongs to the technical field of biological cell extraction. Comprising the following steps: S1, sample collection: collecting 5-10 mL of cystic fluid of a patient with macrocystic lymphangial malformation, and placing the collected cystic fluid in a sterile container; s2, primary centrifugation: centrifuging at a low speed of 1000rpm for 10 minutes, and retaining cell precipitates; s3, red blood cell lysis: adding a red blood cell lysis solution, placing at 4 DEG C for 3-5 minutes, centrifuging, and washing and purifying with PBS (Phosphate Buffer Solution); s4, cell culture: resuspending the cells in a culture medium containing an endothelial growth factor replenishing liquid, culturing at 37 DEG C in 5% CO2, and changing the liquid for 24 hours to remove non-adherent cells; and S5, cell identification: carrying out immunofluorescence staining identification 48-72 hours after cell adherence. The method is simple, convenient and efficient to operate, does not need complex digestion, can enrich high-purity and high-activity lymphatic endothelial cells, provides a stable cell model for related pathogenesis research and drug screening, and has important scientific research and clinical value.

The present invention provides enrichment, detection methods and applications of GPC-3 complexes in blood samples. The enrichment method includes A. collecting peripheral blood from liver cancer patients, placing it in an anticoagulant tube and mixing the whole blood; B. adding multiple volumes of red blood cell lysis solution, pipetting and mixing, lysing on ice, centrifuging and discarding the red supernatant. If the cell pellet is still red, repeat this step; C. washing the pellet 1-2 times, adding PBS or normal saline to resuspend the pellet, centrifuging and discarding the supernatant; D. counting white blood cells, adding PBS to resuspend the cell pellet, taking an appropriate amount to fill the pool, letting it stand for 2-3 minutes, and manually counting the white blood cells using the four large squares in the four corners of the low-power microscope. The calculation formula is as follows: WBC / L=N / 4*10 5 *10 4 , and calculate the number of white blood cells per milliliter of the original suspension, WBC / ml original suspension = N / 4*10 4 *Dilution factor; E. Add lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor to the obtained cells, refrigerate and let stand for a certain period of time, and then centrifuge to obtain the supernatant as the sample to be tested to achieve GPC-3 enrichment.

The invention discloses a pig bone marrow cell preparation method and a pig bone marrow cell preparation system, and relates to the technical field of cell preparation.The pig bone marrow cell preparation method comprises the following steps that pig bone tissue is obtained, under the condition of flowing cell washing liquid, the pig bone tissue is extruded, and the pig bone marrow cells are obtained; the content of the pig bone tissue is released into the flowing cell washing liquid, and pig bone marrow cell mixed liquid is obtained; filtering and centrifuging the pig bone marrow cell mixed solution, and taking precipitate; resuspending the precipitate to obtain a resuspension solution; and mixing the resuspension with a red blood cell lysis solution, washing, centrifuging, and taking the precipitate for cell culture to obtain the pig bone marrow cells. The preparation method disclosed by the invention is relatively simple in operation method, relatively low in cell pollution rate, relatively stable in cell preparation amount, relatively high in cell preparation amount, relatively short in operation time and relatively high in preparation efficiency.

The invention discloses a human whole blood genome DNA extraction kit, an extraction method and application. The DNA extraction kit comprises a red blood cell lysis solution and a DNA extraction solution, the red blood cell lysis solution comprises sucrose, NP40, CA-630, Proclin300, sodium chloride and Tris-HCl, and a solvent is purified water; the DNA extracting solution is prepared from SDS (Sodium Dodecyl Sulfate), EDTA (Ethylene Diamine Tetraacetic Acid), triton X-100, NaOH, Chelex-100, Brij58, Proclin300, trehalose and glycerol, and a solvent is purified water. The invention further discloses a method for extracting the human whole blood genome and application. According to the method, common PCR inhibitors can be effectively removed; the extracted DNA is high in purity, and the product can be directly applied to downstream applications of qPCR, STR, NGS and the like; the requirement on the initial sample size is low; organic solvents such as phenol chloroform are not used, so that the method is more environment-friendly.

The invention relates to the technical field of biomedicine, in particular to a trace blood DNA (deoxyribonucleic acid) extraction method which comprises the following steps: S1, mixing a blood sample and lysate according to a ratio of 1: 3, uniformly mixing by vortex, and centrifuging; s2, protease K is added, vortex mixing is carried out, incubation is carried out at 55 DEG C for 15 min, centrifugation is carried out for 30 s, and 500 microliters of a binding solution is added; and S3, completely transferring the liquid into a purification column with a collection tube, and standing for 2 minutes. The extraction method of the trace blood DNA comprises the following steps: denaturating protein through SDS (Sodium Dodecyl Sulfate), and separating nucleic acid; the binding liquid adopts high salt and low PH, so that the long fragment nucleic acid can be more easily bound on the adsorption column, guanidine hydrochloride can enhance the splitting effect, and the nucleic acid in the blood sample can be fully released due to the dual splitting effect; and on the other hand, the key point is that the step of removing red blood cells by red blood cell lysate is avoided, but the whole blood is directly lysed, so that the loss of white blood cells is reduced, the operation steps are simplified, and the method is suitable for extracting fresh and frozen anticoagulant blood samples.

The invention relates to the technical field of cell engineering, in particular to a placenta macrophage purification and culture method. Comprising the following steps: (1) cutting placenta tissues into pieces, adding an enzymatic hydrolysate for digestion, adding a red blood cell lysis buffer for ice bath, and adding a basic culture medium for resuspending cells to obtain a resuspended cell mixed solution; (2) adding the resuspended cell mixed solution to the upper layer of the lymphocyte separation solution, centrifuging, and sucking out macrophages to obtain purified macrophages; and (3) inoculating the purified macrophages into an amplification culture medium, and culturing to obtain the macrophages subjected to amplification culture. The invention provides an efficient and stable placenta macrophage purification and culture method, through optimized enzymolysis separation, gradient centrifugal purification and a specific amplification culture medium formula, the purity, the multiplication capacity and the functional stability of the macrophage are remarkably improved, and the placenta macrophage purification and culture method has a practical application value.

The invention discloses a preparation method of a traditional Chinese medicine polysaccharide assisted pancreatic cancer DC vaccine. The preparation method comprises the following steps: extracting mouse bone marrow dendritic cells; the preparation method comprises the following steps: cracking and centrifuging mouse bone marrow dendritic cells through a red blood cell lysis solution, cleaning a culture medium, and culturing in a special culture medium to obtain immature dendritic cells; collecting the PANC2 pancreatic cancer cells subjected to amplification culture, counting, putting into a cryopreservation tube, and preparing a tumor antigen by a low-temperature and normal-temperature repeated freezing and thawing method; the preparation method comprises the following steps: co-culturing immature dendritic cells and tumor antigen for 12-24 hours, adding traditional Chinese medicine polysaccharide, continuously culturing until the dendritic cells are mature, and re-suspending the co-cultured cells in normal saline to obtain the pancreatic cancer DC vaccine. The prepared traditional Chinese medicine polysaccharide assisted pancreatic cancer DC vaccine has the advantages of high maturity, enhanced phenotypic signal molecule expression and the like, and can effectively inhibit tumor cell growth when being independently applied to a pancreatic cancer tumor-bearing mouse model.

The invention relates to the field of urine sample detection, in particular to a urine analysis and urine sample detection method which comprises the following steps: urine sampling, physical analysis, chemical detection and microscope detection. According to the invention, a worker can observe the number of white blood cells and the number of red blood cells in a urine sample through a high-power microscope, the red blood cells in the sample 1 are removed through ACK red blood cell lysis buffer, and the white blood cells in the sample 3 are removed through centrifugation and filtration; therefore, a worker can compare the number of the white blood cells in the sample 2 with the number of the white blood cells in the sample 1 and compare the number of the red blood cells in the sample 2 with the number of the red blood cells in the sample 3, so that the number of the white blood cells and the number of the red blood cells in the sample 2 can be rechecked; the detection accuracy of the number of white blood cells and the number of red blood cells in the urine sample is further improved.

The application provides a preparation method and application of a liver tumor tissue single-cell suspension, and the preparation method comprises the following steps: step 1, pretreating liver tumor tissue; step 2, step-by-step enzymolysis; the pretreated liver tumor tissue in step 1 is subjected to step-by-step enzymolysis by using an enzyme solution, and the enzyme solution in each step is collected after filtration to obtain a cell suspension; step 3, gradient centrifugation; the cell suspension is subjected to gradient centrifugation by using a cell separation solution to separate immune cells and liver parenchymal cells; step 4, red blood cell lysis; the immune cells obtained in step 3 are subjected to red blood cell lysis by using a red blood cell lysis solution, and centrifugal washing is carried out to obtain an immune cell precipitate; step 5, collecting the liver parenchymal cells in step 3 and the immune cell precipitate in step 4, washing the liver parenchymal cells and the immune cell precipitate by using a cell washing solution, centrifuging to obtain a cell precipitate, resuspending the cell precipitate by using a cell resuspension solution, and filtering to obtain the liver tumor tissue single-cell suspension.

The invention relates to the technical field of biological medicine, and provides a peripheral blood crack red method for efficiently capturing neutrophil through single cell sequencing. Comprising the following steps: collecting human peripheral blood into an ethylenediaminetetraacetic acid-containing anticoagulant tube; the method comprises the following steps: taking ethylenediamine tetraacetic acid anticoagulant whole blood, adding a red blood cell lysis buffer, carrying out lysis at room temperature, waiting for 1 minute after a turbid mixed blood solution becomes clear and transparent, adding an FACS cell resuspension to terminate lysis red, and carrying out centrifugation; adding an FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding an FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; and adding a phosphate buffer solution to slowly resuspend the cells, and centrifuging. According to the method disclosed by the invention, the high activity and low cell debris rate of the neutrophile granulocyte can be ensured by selecting the proper anticoagulant, accurately controlling the erythrocyte lysis condition and careful cell resuspension and centrifugal operation, a proper single-cell suspension is provided for single-cell RNA sequencing, and the analysis of the characteristics and functions of various subgroups of the neutrophile granulocyte is facilitated.

The present invention discloses a dissociation kit for mouse colorectal single cell suspension, which is composed of the following components: a first enzymatic digestion solution, a second enzymatic digestion solution, a third enzymatic digestion solution, a PBS buffer solution, an RPMI-1640 culture medium, and an erythrocyte lysate; the first enzymatic digestion solution is a 20 mM EDTA solution; the second enzymatic digestion solution is a 0.25% trypsin-EDTA solution; the concentration of neutral protease in the third enzymatic digestion solution is 100 mg / mL, the concentration of collagenase I is 100 mg / mL, and the concentration of collagenase II is 100 mg / mL; a method for extracting mouse colorectal single cell suspension using the dissociation kit is also disclosed. The reagent materials used in the dissociation solution in the kit are easy to obtain, contain no harmful components, are safe and environmentally friendly, the method is simple to operate, and the final result after the cooperation of each step has high repeatability and success rate, and can stably obtain a mouse colorectal single cell suspension with high viability and low impurities.

The present invention discloses a kit for detecting multiple cell subsets, which kit comprises two sets of small molecule antibodies conjugated with different fluorescent groups, a reaction buffer and a red blood cell lysate. The antibodies include CD3 molecule labeled with APC / CY7, CD4 molecule labeled with BV605, CD8 molecule labeled with Alexa700, HLA-DR molecule labeled with Percp / Cy5.5, CD45RA molecule labeled with APC, CD19 molecule labeled with FITC, CD197 molecule labeled with BB700, CD16 molecule labeled with APC and CD56 molecule labeled with PE. Among them, the combination of CD3, CD19, CD56, CD16 and HLA-DR fluorescent antibodies is set B, and the combination of CD3, CD4, CD8, CD45RA and CD197 fluorescent antibodies is set T. The present invention also provides the use of the kit for detecting multiple cell subsets.

This invention belongs to the field of biomedical technology, specifically relating to a method and kit for improving the sensitivity and specificity of circulating tumor cell (CTC) detection. The method first treats anticoagulated blood with erythrocyte lysis buffer, centrifuges to obtain a cell suspension (including leukocytes and circulating tumor cells); then, a radiosensitizer is added and the suspension is irradiated; next, single-cell gel electrophoresis is used to detect the degree of DNA damage in the test cells; finally, leukocytes and tumor cells are distinguished based on cell morphology using a fluorescence microscope or fluorescence scanner. This invention is the first to detect the number and type of circulating tumor cells (CTCs) in peripheral blood based on ionizing radiation and radiosensitization effects, according to the degree of DNA damage in tumor cells. Compared with existing technologies, this detection method and kit are not only simple and rapid, but also significantly improve detection sensitivity and specificity. Furthermore, it is independent of the physical properties of cells and tumor cell surface markers, requires a small sample volume, is highly versatile, and can detect CTCs in different types of solid tumors.

The invention discloses a spleen neutrophil detection kit and a detection method thereof, and relates to the technical field of biological detection.The spleen neutrophil detection kit specifically comprises a sample treatment reagent, a cell separation reagent, a detection recognition reagent, a dyeing auxiliary reagent and a detection auxiliary reagent, collagen is specifically decomposed and free DNA is degraded through enzyme digestion, cell aggregation is prevented, and the detection accuracy is improved. In this way, efficient dissociation of spleen tissue is achieved, cell damage is reduced while a single-cell suspension is obtained, selective hypotonic lysis of red blood cells is achieved through a red blood cell lysis solution, white blood cells and neutrophils are reserved, the red blood cells are classified, the phenomenon that the red blood cells interfere with subsequent detection is avoided, and the detection accuracy is improved. Meanwhile, the density difference between the neutrophile granulocytes and other leukocytes is utilized, rapid separation is achieved in a centrifugal layering mode, the neutrophile granulocytes can be concentrated on an interface layer, separation, quantification and identification of the spleen neutrophile granulocytes are achieved, the initial enrichment efficiency is improved, and the reliability of subsequent detection results is ensured.

The invention relates to the technical field of hepatocellular carcinoma detection, and discloses a postoperative circulating tumor cell detection method based on hepatocellular carcinoma. The method comprises the following steps: collecting a venous blood sample within half an hour after a hepatocellular carcinoma patient, treating the venous blood sample with a red blood cell lysis solution to remove red blood cell components, and performing centrifugal separation to obtain a cell precipitate; embedding the precipitate in a paraffin matrix to form a cell paraffin block, and continuously slicing to obtain a slice sample to be detected; labeling surface antigens of the circulating tumor cells in the slices through specific antibodies by adopting an immunohistochemical technology; collecting a microscopic image of the marked sample, and extracting morphological characteristic parameters and dyeing intensity distribution data related to the circulating tumor cells; a recognition model is constructed based on the data, and positive circulating tumor cells and background interference signals are distinguished. The method is standard in process, achieves efficient detection of circulating tumor cells, and meets clinical requirements of hepatocellular carcinoma postoperative monitoring.