37 results about "Erythrocyte lysis" patented technology

The invention relates to an integrated microfluidic chip for capture of cancer cells in whole blood. The integrated microfluidic chip for capture of cancer cells in whole blood comprises three inlets, an erythrocyte lysis unit, a leucocyte capture unit, a cancer cell capture unit and an outlet, wherein the three inlets, the erythrocyte lysis unit, the leucocyte capture unit, the cancer cell capture unit and the outlet are connected orderly. The leucocyte capture unit comprises a first diverter, a soft magnetic microcolumn array and permanent magnets. The first diverter is connected with the soft magnetic microcolumn array. The permanent magnets are arranged at two sides of the soft magnetic microcolumn array. The cancer cell capture unit comprises a second diverter and a capture microstructure array, wherein the second diverter and the capture microstructure array are connected orderly. Compared with the prior art, the integrated microfluidic chip for capture of cancer cells in whole blood has the advantages of high integration degree, simple operation, high cell capture efficiency, simple manufacture processes and the like.

The invention provides an erythrocyte lysis buffer. The erythrocyte lysis buffer comprises the following components: 4.5-5.5 mmol/L of sodium chloride, 0.8-1.3% (v/v) of triton 100, 300-340mmol/L of glucose and 0.008-0.012 mol/L of trishydroxymethyl aminomethane. The erythrocyte lysis buffer can effectively lyse the erythrocyte in a whole blood sample, separate and enrich leucocyte in the blood, and facilitate the subsequent nucleic acid extraction operation of the leucocyte, so as to obtain the leucocyte DNA in the whole blood sample and the DNA of virus in the leucocyte.

The invention provides a mammal blood genome DNA extraction kit which is composed of an erythrocyte lysis buffer, a leukocyte lysis buffer, a protein precipitation liquid and DNA precipitation liquid. Compared with the prior art, the mammal blood genome DNA extraction kit has the following technical effects: (1) no organic reagent is used, and the mammal blood genome DNA extraction kit is friendly to the environment and harmless to a human body; (2) DNA with relatively high concentration and purity can be obtained, and stretches of DNA are preserved; (3) one-step removing method is used, and is simple to operate and applicable to a wide crowd; (4) the operation time is short, a DNA sample needed by an experiment can be obtained in a short time, so that the efficiency of the experiment is greatly improved; and (5) the cost is low and the price is low.

Preparation and detection methods of an animal heart single-cell suspension are disclosed. The preparation method includes 1) preparing an animal heart ischemia reperfusion model; 2) preparing an animal isolated hear tissue, flushing the tissue to remove blood, adding heart digestive juice, cutting the tissue into pieces, fully mixing the tissue pieces with the digestive juice and performing digestion; and 3) filtering a mixture solution obtained in the step 2), performing centrifugation, removing a supernatant liquid, terminating digestion, adding a phosphate buffer liquid for suspension, adding an erythrocyte lysis solution, performing centrifugation, removing a supernatant liquid, and adding a phosphate buffer liquid for suspension to obtain the animal heart single-cell suspension. Thepreparation method can reduce destroy to cells, and the single-cell suspension preparation method which is reliable and stable is provided. The number of cells in the prepared suspension is high. Thedetection method is simple to operate and comprehensive in data.

A lab-on-a-chip (LOC) device for pathogen detection, genetic analysis and proteomic analysis of a biological sample, the LOC device having an inlet for receiving the sample, a supporting substrate, a leukocyte dialysis section for dividing the sample into a leukocyte stream, and a pathogen and erythrocyte stream containing cells and sample constituents smaller than a first size threshold, a pathogen dialysis section for dividing the pathogen and erythrocyte stream into an erythrocyte stream and a pathogen stream containing cells and sample constituents smaller than a second size threshold, a leukocyte lysis section downstream of the leukocyte dialysis section for lysing the leukocytes with a lysis reagent to release genetic material and proteins therein, a leukocyte incubation section downstream of the leukocyte lysis section for enzymatic reaction of the genetic material with enzymes, a pathogen lysis section downstream of the pathogen dialysis section for lysing the pathogens with a lysis reagent to release genetic material and proteins therein, a pathogen incubation section downstream of the pathogen lysis section for enzymatic reaction of the genetic material with enzymes, an erythrocyte lysis section downstream of the pathogen dialysis section for lysing the erythrocytes with a lysis reagent to release proteins therein, a first leukocyte nucleic acid amplification section downstream of the leukocyte incubation section for amplifying nucleic acid sequences, a second leukocyte nucleic acid amplification section downstream of the first leukocyte nucleic acid amplification section for amplifying nucleic acid sequences, a first pathogen nucleic acid amplification section downstream of the pathogen incubation section for amplifying nucleic acid sequences, and, a second pathogen nucleic acid amplification section downstream of the first pathogen nucleic acid amplification for amplifying nucleic acid sequences, wherein, the leukocyte dialysis section, the pathogen dialysis section, the leukocyte lysis section, the pathogen lysis section, the leukocyte incubation section, the pathogen incubation section, the first and second leukocyte nucleic acid amplification sections and the first and second pathogen nucleic acid amplification sections are all supported on the supporting substrate.

The invention discloses a kit for rapidly capturing, releasing and detecting circulating tumor cells without damage, and relates to the field of biotechnology and biomedicine. The kit includes a CTCsmagnetic nano capture probe solution, an erythrocyte lysis solution, a CTCs releasing solution, a CTCs detecting solution and washing buffer; CTCs magnetic nano capture probes are the magnetic nanoparticles whose surfaces are modified with folic acid; the folic acid on the CTCs magnetic nano capture probes specifically bind to the folate receptors on the surfaces of the circulating tumor cells, sothat the capturing and separation of the circulating tumor cells can be realized at an external magnetic field; and the CTCs detecting solution can perform fluorescence labeling on the released CTCs,so that observation and counting can be realized under a fluorescence microscope. The CTCs captured and released by the kit retain very high activity, so that the kit has significant meaning on the early in vitro screening and diagnosis of cancer and the follow-up study of the CTCs; and the components in the kit can be selectively used according to specific demands, so that simple and convenientoperation can be realized, and therefore, the kit can have wide application prospects.

A lab-on-a-chip (LOC) device for pathogen detection, genetic analysis and proteomic analysis of a biological sample, the LOC device having an inlet for receiving the sample, a supporting substrate, a leukocyte dialysis section for dividing the sample into a leukocyte stream, and a pathogen and erythrocyte stream containing cells and sample constituents smaller than a first size threshold, a pathogen dialysis section for dividing the pathogen and erythrocyte stream into an erythrocyte stream and a pathogen stream containing cells and sample constituents smaller than a second size threshold, a leukocyte lysis section downstream of the leukocyte dialysis section for lysing the leukocytes with a lysis reagent to release genetic material and proteins therein, a leukocyte incubation section downstream of the leukocyte lysis section for enzymatic reaction of the genetic material with enzymes, a leukocyte nucleic acid amplification section downstream of the leukocyte incubation section for amplifying nucleic acid sequences, a pathogen lysis section downstream of the pathogen dialysis section for lysing the pathogens with a lysis reagent to release genetic material and proteins therein, a pathogen incubation section downstream of the pathogen lysis section for enzymatic reaction of the genetic material with enzymes, a pathogen nucleic acid amplification section downstream of the pathogen incubation section for amplifying nucleic acid sequences, an erythrocyte lysis section downstream of the pathogen dialysis section for lysing the erythrocytes with a lysis reagent to release proteins therein, wherein, the leukocyte dialysis section, the pathogen dialysis section, the leukocyte lysis section, the pathogen lysis section, the erythrocyte lysis section, the leukocyte incubation section, the pathogen incubation section, the leukocyte nucleic acid amplification section and the pathogen nucleic acid amplification section are all supported on the supporting substrate.

The invention relates to an alpha fetoprotein kit based on an aptamer fluorescent probe AFP4, a method for determining the alpha fetoprotein AFP concentration and compositions and ingredients of a determining reagent and belongs to the technical field of medical examination determination. The kit provided by the invention mainly comprises an erythrocyte lysis solution, a phosphate buffer PBS, an alpha fetoprotein AFP standard substance and an alpha fetoprotein AFP aptamer fluorescent probe. The concentration of the alpha fetoprotein AFP can be measured through blood sample splitting decomposition and mixing spawn raising treatment as well as fluorescence spectrophotometer detection. The alpha fetoprotein kit provided by the invention has the advantages of simple sample treatment, convenience in operation, short detection time, strong detection specificity, high sensitivity, high repeatability of detection results, and the like.

The invention relates to a pretreatment reagent of a sample for leukocyte classification. The pretreatment reagent contains a hemolytic agent, and an active component of the hemolytic agent is mainlycomposed of organic acid and a polymer non-ionic surfactant; the organic acid selects from at least one of formic acid, acetic acid and propionic acid; the polymer non-ionic surfactant selects from atleast one of polyvinylpyrrolidone and polyethylene glycol. When the pretreatment reagent of the sample is used for treating the sample for the leukocyte classification, the erythrocyte lysis efficiency is high, the leukocyte destructiveness is small, and the treated sample is long in preservation time.

The invention discloses a method for isolating dendritic cells, which comprises the following steps: (1) preparation of bone marrow mononuclear cell suspension: taking bone, cutting the epiphysis, washing the middle bone to obtain bone marrow fluid, and cutting the epiphysis until it becomes viscous mixed with the aforementioned bone marrow fluid, placed on a strainer, washed 3 to 4 times to obtain a cell suspension, centrifuged, collected the precipitate, added red blood cell lysate, left standing, centrifuged, washed, and obtained bone marrow mononuclear cells; (2) imDC Cell culture: the bone marrow mononuclear cells obtained in step (1) were resuspended in medium 1, inoculated and cultured, centrifuged, cultured in medium 2 added with growth factors, and cultured continuously for 7 days to obtain imDC cells; (3) mDC Cell culture: Add cell culture medium 3 containing maturation-promoting factors to imDC cells to continue induction culture. The method for isolating dendritic cells of the present invention can effectively separate dendritic cells from mouse bone marrow, and has excellent clinical application prospects.

The invention discloses a kit for in-vitro separation purification of karyocytes and removal of erythrocytes. The kit comprises a precipitation solution, a purification solution, a suspension solution and an erythrocyte lysis solution, wherein the precipitation solution, the purification solution, the suspension solution and the erythrocyte lysis solution are separatedly packaged. The precipitation solution is composed of normal saline and hydroxyethyl starch dissolved in the normal saline and has a weight volume concentration of 6%. The purification solution is a mixture of polysucrose and sodium diatrizoate. The suspension solution is normal saline. The erythrocyte lysis solution comprises a stock solution 1 and a stock solution 2, wherein the stock solution 1 and the stock solution 2 are separatedly packaged; the stock solution 1 comprises 10g/L of glucose, 0.6g/L of potassium dihydrogen phosphate, 3.58g/L of Na2HPO4.7H2O and 0.1g/L of a phenol red solution; and the stock solution 2 comprises 1.86g/L of CaCl2.2H2O, 4.0g/L of potassium chloride, 80g/L of sodium chloride, 1.04g/L of magnesium chloride and 2.0g/L of MgSO4.7H2O. The invention also provides a use method of the kit.

The invention discloses human glioma primary cells and an in-vitro isolation and culture method and an application thereof. The cells are named as a low-grade human glioma primary cell XHLG-07 and a high-grade human glioma primary cell XHHG-02 and have the preservation numbers of CCTCC NO:C2018256 and CCTCC NO:C2018215 respectively. The isolation and culture method comprises the steps: acquiring aglioma tissue excised after operation in clinic in vitro, removing blood vessels in the tissue and an adipose tissue by anatomical tweezers, digesting by collagenase/pancreatin, filtering, carrying out low-speed centrifugation to obtain a cell precipitate, carrying out lysis of erythrocytes with an erythrocyte lysis solution, resuspending cells in a DF31 complete culture medium and carrying out inoculation culture. The isolation method can be used for acquiring WHO glioma primary cells with different grades. The obtained cells can be used for analyzing pathogenesis, drug sensitivity and metastasis of human brain gliomas with different grades in vitro and in vivo, and provide research materials more similar to the biological characteristics of clinical tumors for the study of human brain gliomas with different grades.

The invention relates to a cell preservation solution for rapid cell immunohistochemistry as well as a preparation method and application of the cell preservation solution. The cell preservation solution provided by the invention is mainly composed of a buffer solution, an erythrocyte lysis reagent, an osmotic pressure maintaining agent, a cell morphology maintaining reagent, a fixing agent, a preservative, a mucus treatment reagent, an anti-aggregation reagent and a reagent for cell immunohistochemical cell antigen treatment. The reagent for cell immunohistochemical cell antigen treatment comprises formalin and zinc salicylate, and the formalin and the zinc salicylate cooperate with each other to effectively catalyze an immunohistochemical reaction, so that cell antigens do not need to berepaired, the reaction time of cell immunohistochemical detection is shortened, the detection efficiency is improved, and the timeliness of pathological diagnosis is enhanced. The cell preservation solution provided by the invention can be applied to preserving or collecting various cell samples such as cervical exfoliated cells, pleural effusion, alveolar lavage fluid, cerebrospinal fluid, sputum and urine. Meanwhile, the invention provides a preparation method of the cell preservation solution .

The invention discloses a kit for genome DNA extraction and an application of the kit. The kit comprises a lysis solution and an erythrocyte lysis solution, wherein the lysis solution contains 20 to 300mM of Tris-HCl, 20 to 100mM of EDTA, 0.3 to 2M of NaCl, 0.5 to 5 percent of PEG8000 and 0.5 to 10 percent of Triton X-100, and SDS or Tween 20; the erythrocyte lysis solution contains 10 to 100mM ofTris-HCl, 1 to 50mM of EDTA, 10 to 300mM of NaCl and 0.01 to 0.2 percent of Triton X-100. According to the kit, through optimization of the lysis solution and the erythrocyte lysis solution, polysaccharide can be effectively removed, phenol is combined, phenol is prevented from being combined with DNA, the problems of viscosity and turbidity are solved, the kit is suitable for various different samples, and the purity and yield of extracted genome DNA can meet various downstream experiment use requirements.

The invention discloses a sorting method of human osteoblasts, which comprises the following steps: performing digestion twice by using endotoxin-free collagenase with the digestion concentration of 1.0 mg/m, cell suspension centrifugal force being 400; after the twice digestion is finished, filtering out bone fragments by using a 70-micron filter screen, collecting the supernatant, adding PBS to50 ml, and centrifuging the liquid; carrying out lysis for multiple times by using an erythrocyte lysis solution, wherein the lysis cannot last for excessive long time, until no obvious red substanceexists in cell precipitate; filtering the liquid by using a 40-micron filter screen to obtain a supernatant after the erythrocyte splitting is finished; and washing the red-split filtered cells with PBS (phosphate buffer solution) for multiple times, centrifuging the liquid, and removing impurities. The FACS sorting employs ALPL and CD45/34/31 antibodie for labeling and separating human osteoblasts, so that the activity of the human osteoblasts obtained through the method is higher, the number of cells is larger, and cell impurities are greatly reduced.

The present invention provides a circulating tumor cell separation and enrichment kit. The circulating tumor cell separation and enrichment kit comprises a circulating tumor cell separation and enrichment preservation solution, and the circulating tumor cell separation and enrichment preservation solution is an aqueous solution containing the following components by concentration of 3-4 mmol/L ofNaCl, 12-14 g/L of NH4Cl, 11.5-14 mmol/L of Tris-HCl, 29-31.5 mmol/L of 4-hydroxyethyl piperazine ethanesulfonic acid, and 0.03-0.06 w/v% of corn oligopeptides. The circulating tumor cell separation and enrichment preservation solution can effectively preserve CTCs in a biological sample, keep the surface and cell internal antigen activity of the CTCs, improve the enrichment efficiency of the CTCsin the sample to be detected, has a very good erythrocyte lysis effect, and can realize normal-temperature storage or transportation of the CTCs-containing biological sample.

The invention provides a method for reconstructing a colorectal cancer organoid immune microenvironment. The method comprises the following steps: constructing a colorectal cancer organoid by using fresh colorectal cancer tissue; obtaining peripheral blood, then performing erythrocyte lysis, and separating lymphocytes by adopting a lymphocyte separating medium; enriching CD3 <+> T cells; and digesting the organoids into single cells, co-culturing the single cells and the cultured CD3 <+> T cells according to a ratio of 1: (10-30), and constructing the colorectal cancer patient-derived organoids immune microenvironment according to the steps. According to the method for reconstructing the colorectal cancer organoid immune microenvironment provided by the invention, fresh colorectal cancer tissue from the same individual and CD3 <+> T cells from peripheral blood are co-cultured to obtain the organoid immune microenvironment, it is, from cellular morphology and ATP values, shown that immune cells have an obvious killing effect on organoids of CRC patients, and an in-vitro experimental basis is provided for immunotherapy of clinical autologous T cell transfusion.

The invention discloses a rat macrophage culture method. The method comprises the following steps: killing young SD rats which are born for 3 weeks by a neck removal method, soaking the rats in 75% alcohol for disinfection for 3 minutes, transferring the rats into an ultra-clean workbench for operation after disinfection is completed, cutting posterior limb skin, removing leg muscles, exposing thighbones, stripping external muscles of the thighbones cleanly, and cutting off the thighbones; cutting off femoral heads at the two ends of the thighbones, sucking a certain amount of MEM-alpha basalculture medium liquid by using an injector, blowing out bone marrow, and flushing the bone marrow cleanly in the blowing process; blowing and beating the bone marrow for 30 times by using a pasteur pipette to prepare a single-cell suspension, performing centrifuging by using a centrifuge, collecting cells, and discarding the supernatant; and adding erythrocyte lysate. According to the invention, the rat bone marrow is subjected to primary centrifugal separation, erythrocyte lysis, secondary centrifugal separation and culture by using a special culture medium for macrophages, so that the cultured macrophages are relatively high in purity, the culture speed is relative high, and the research requirements are met.

There is provided a method of inducing pluripotency in a hematopoietic cell. The method comprises providing a blood sample that has been collected from a subject in the absence of any polysaccharide preparation used for separating blood components. The sample is depleted of red blood cells by treating the sample with a hypotonic erythrocyte lysis buffer, in the absence of any polysaccharide preparation used for separating blood components, thus obtaining a remaining cell population. The remaining cell population in the sample is expanded in a hematopoietic expansion medium to obtain an expanded cell population that contains CD71+ cells, and the expanded cell population containing the CD71+ cells is then cultured in the presence of Oct4, Sox2, and Klf4, and optionally c-MYC, in human embryonic stem cell medium to induce pluripotency.

The invention discloses a bone marrow peptide extraction and preparation method, which comprises: taking a fresh bone, removing connective tissue, filling the bone marrow into a centrifugation vessel, carrying out ultrasonic treatment on the treated solution to break cells, carrying out centrifugation to remove the supernatant, adding an erythrocyte lysis buffer and a sterile PBS buffer solution, and carrying out centrifugation to obtain the bone marrow peptide. And performing continuous graded gradient ultrafiltration on the mixed solution to prepare a concentrated raw material solution, and preparing different preparations of the bone marrow peptide according to the concentration requirement. According to the method, bone marrow peptide extraction can be effectively carried out on various materials. Meanwhile, the extraction rate of bone marrow peptide is greatly increased, the activity of bone marrow peptide can be reserved through ultrasonication, the denaturation trend of protein is effectively avoided. Meanwhile, the production time is shortened; the red blood cell lysis buffer is matched with the PBS buffer solution, so that the generation rate of the peptide can be further improved, the bone marrow peptide with the highest quality can be obtained under the filtration of an ultrafilter with an ultrafiltration membrane, and the influence of the molecular weight distribution of the polypeptide can be reduced through the spray drying process and the control of the temperature of inlet air and outlet air.