109 results about "Blood lymphocyte" patented technology

A composition and method for cryopreserving cells in a non-toxic medium is described. The composition and method provide cells for cell therapy and other in vivo applications.

The invention relates to a method for culturing an autologous peripheral blood lymphocyte. The method comprises the following steps: (1) separating a mononuclear cell from peripheral blood, resuspending in an X-VIVO15 serum-free culture medium to obtain cell concentration of 1*10<6> / mL, and culturing for 3 days; (2) supplementing the X-VIVO15 serum-free culture medium to 100 mL, adding IL-21*10<3> U / mL, and culturing for 1 day; (3) supplementing the X-VIVO15 serum-free culture medium to 200-240 mL, adding IL-21*10<3> U / mL, and culturing for 3 days; (4) supplementing the X-VIVO15 serum-free culture medium to 1000 mL, adding IL-21*10<3> U / mL, CTLA-4mAb100n g / mL and PD-1mAb100n g / mL; and (5) culturing for 5-7 days to prepare the autologous peripheral blood lymphocyte. The method disclosed by the invention can be used for improving the activation efficiency and amplification efficiency of an effector cell group by adding multiple monoclonal antibodies and cell factors to the X-VIVO15 serum-free culture medium, and can be used for effectively reducing the content of T regulatory cells by covering CTLA-4 and PD-1 molecules of the surfaces of all CIK cells by loading CTLA-4 and PD-1 antibodies in vitro especially, thus further enhancing the killing effect of the CIK cells on tumors.

This invention discloses a lymphocyte separation tube, which comprises a tube body. The bottom of the tube body is set loose and porous material. This invention also discloses a method for separating blood lymphocyte by utilizing the lymphocyte separation tube. The lymphocyte separation tube has such advantages as convenient utilization, simple preparation process, and high safety,and no toxicity.

The invention specifically relates to a method for extracting immune cells from marrow, belonging to the technical field of cytobiology. The method comprises the following steps: (1) acquiring human marrow, diluting the human marrow with an alpha-MEM nutrient solution containing fetal calf serum, then carrying out centrifugation and discarding supernatant and a fat layer so as to obtain marrow cells; (2) transferring human peripheral blood into a centrifuge tube, carrying out centrifugation, sucking up blood plasma at an upper layer for subsequent usage and cells at a lower layer for separation of peripheral blood lymphocytes, and adding an alpha-MEM nutrient solution containing the blood plasma at the upper layer into the marrow cells to prepare a cell suspension; (3) adding a separating medium into another centrifuge tube, flatly spreading the cell suspension onto the separating medium, carrying out centrifugation, and sucking up albuginea-layer cells so as to obtain immune cells; and (4) adding isopyknic immune cells into an immune cell cryopreservation solution, carrying out uniform mixing and carrying out immune cell cryopreservation. The method provided by the invention can effectively improve the separation rate of the immune cells and the survival rate of the immune cells after cell cryopreservation and enhances the security of cells transfused back to a patient.

The invention relates to a method for culturing autologous peripheral blood lymphocytes, comprising the following steps of: (1) separating a PBMC (Peripheral Blood Mononuclear Cell) from peripheral blood, then heavily suspending in a serum-free culture medium, and statically culturing to prepare a primary culture solution; (2) replenishing the serum-free culture medium to the primary culture solution, simultaneously adding IL (Interleukin)-2, and statically culturing to prepare a secondary culture solution; (3) replenishing the serum-free culture medium to the secondary culture solution, simultaneously adding IL-2, and statically culturing to prepare a culture solution; and (4) uniformly dividing the culture solution into two parts, adding the serum-free culture medium to complement a sufficient volume, adding IL-2, statically culturing, and repeating the step (4) to prepare the autologous peripheral blood lymphocytes. As multiple monoclonal antibodies and cell factors are added to the serum-free culture medium, the activation efficiency and the amplification efficiency of effector cell masses are improved. Through detection, the activation efficiency and the amplification efficiency are obviously improved as compared with the existing method, and a large amount of cell masses occur after the effector cell masses are activated about 3 days.

The invention relates to a traditional Chinese medicine immunopotentiator (an astragalus polysaccharide immunopotentiator for short) prepared from astragalus extracts and sulfated epimedium polysaccharides, which belongs to the field of immunological adjuvants of livestock and poultry. 1,000ml of the liquid medicine is prepared from 40g of astragalus and 120g of epimedium herb. The preparation method of the immunopotentiator comprises the following steps of: decocting the astragalus with water for three times, then merging the obtained filter liquor and condensing the filter liquor into 500ml of astragalus solution; extracting the epimedium polysaccharides by using the epimedium herb water decoction and ethanol precipitate method, then decorating the polysaccharides by using the chlorosulfonic acid-pyridine method, and preparing the polysaccharides into 500ml of sulfated epimedium polysaccharide solution after carrying out distilled water dialysis on the polysaccharides; and mixing the astragalus solution and the sulfated epimedium polysaccharide solution, and then filtering, sub-packaging and sterilizing to obtain the traditional Chinese medicine astragalus polysaccharide immunopotentiator. The immune experiment shows that the astragalus polysaccharide immunopotentiator has the advantage of obviously improving the proliferation of peripheral blood lymphocyte of chicken and enhancing the cellular immunity, obviously improving the potency of a serum antibody, promoting the lymphocyte proliferation, enhancing the cellular immunity and humoral immunity, and improving the immune response of a vaccine by coordinately immunizing chickling by using the Newcastle disease vaccine.

The invention belongs to the technical field of immune cells in-vitro culture, and discloses a method for culturing cord blood lymphocyte DC-CIK. According to the invention, cord blood is taken as a processing object, and is subjected to separation of cord blood mononuclear cells, isolated culture of DC cell, induction culture of CIK cell, and co-culture of DC and CIK to prepare the DO-DIK cell, CTLA-4mAb and PD-1mAb are added, and the maturated DO-DIK cell is subjected to centrifugal collection to obtain the product. By using CD3mAb coating culture bottle, continuous stimulation is carried out at an early stage of culture, cytokine usage is reduced, effector cell population activation efficiency and amplification efficiency are increased, and the culture cost is reduced.

The invention relates to a human peripheral blood lymphocytes culture medium and the application thereof. The human peripheral blood lymphocytes culture medium of the invention comprises: RPMI1640 liquid culture medium 77-81.9%, bovine blood 10-14.9%, PHA 4-9%, non-sulfated glycosaminoglycan 2-5%, CPPs 2-5% and Double-resist 0.05-0.1%. The culture medium provided in the invention achieves a high lymphocyte conversion rate, a high lymphocyte split index and a good reagent stability; can obtain a human chromosome figure which can provide a firm basis to chromosome core type analysis and clinic diagnosis.

The invention relates to a botanical drug compound recipe for preventing and treating microwave radiation injury. According to the invention, it is found that botanical drugs mainly consisting of peony root, milkvetch root, duckweed and red sage root can be used for preparing a medicine used for preventing and treating microwave radiation injury. According to results of abundant experiments of detection of learning and memory capacity of rats, hippocampal amino acid neurotransmitters, sperm motility, the rate of teratosperm, myocardial metabolic enzyme (CK-MB, LDH and AST activity) and the content of Ca<2+> in serum of rats, the number of peripheral blood lymphocytes and the like, the medicine provided in the invention has an obvious treatment effect on injury to the brain, reproduction, the heart and immunity of rats caused by microwave radiation, and the effect is superior to that of medicines on the market.

The invention relates to a peripheral blood lymphocyte subpopulation cell concentration detection kit and a detection method thereof, and belongs to the technical field of cell detection and screening. According to the technical scheme, the peripheral blood lymphocyte subpopulation cell concentration detection kit comprises an antibody kit and a stimulation liquid kit which is coordinately used with the antibody kit and is characterized in that a six-hole reagent tank is stored in the antibody kit, reagent tubes ranging from number A to number F and representing six antibody solutions are correspondingly arranged in the six-hole reagent tank according to the numbers of the six-hole reagent tank, and a standby reagent bottle is further stored in the antibody kit; after the detection of peripheral blood lymphocyte subpopulation cell concentration and the detection of CD4+T cell subpopulation cell concentration are carried out, samples are placed on a flow cytometer to analyze flow cytological maps, relative percentage is acquired and analyzed by MultiSET software or CellQuest software, and the cell absolute number of various subpopulations is calculated. The peripheral blood lymphocyte subpopulation cell concentration detection kit has the advantages that the technical problem that cell detection is large in errors and complicated in steps is solved, and the detection kit is capable of providing various antibodies and reagents, capable of simultaneously testing a plurality of samples during clinical application, simple in test steps, good in quality control and high in cell detection precision.

The invention belongs to the field of immunodetection and relates to a detection kit for distinguishing cow pathogenic mycobacteria infection from non-pathogenic mycobacteria infection and a method thereof. The detection reagent comprises combined fusion protein rE6-M63-H70 used as a specific stimulation origin, the combined fusion protein can effectively stimulate sensitized peripheral blood lymphocyte cultured in vitro to release Gamma-interferon (IFN-Gamma) at a high level. The cow IFN-Gamma release test established by using the detection reagent rE6-M63-H70 combined fusion protein as the stimulation origin overcomes the insufficiencies of serology detection method and the IFN-Gamma release test with PPD as the stimulation origin, thus enjoying very high sensitivity and specificity and distinguishing cow pathogenic mycobacteria ( such as mycobacterium bovis) infection from non-pathogenic mycobacteria (such as mycobacterium avium or non-pathogenic mycobacteria) infection and even distinguishing the cow pathogenic mycobacteria infection from BGG immunity; therefore, the detection kit and the method of the invention can be effectively used to detect the clinical cow pathogenic mycobacteria infection.

The invention provides a Canine-SLAM / Vero cell line and a constructing method, wherein the constructing method comprises the following steps of: collecting canine distemper positive canine anticoagulated blood, separating peripheral blood lymphocytes by applying a canine lymphocyte separation liquid, extracting total RNA (Ribonucleic Acid), and obtaining a canine SLAM gene by uisng a PCR (Polymerase Chain Reaction) method; constructing pCAGGS-Neo / SLAM plasmids; transfecting a constructed pCAGGS-Neo / SLAM expression cassette into a Vero cell, and screening the cell line for stably expressing an SLAM by adopting G418; and identifying and detecting the expression condition of the SLAM on the surface of a cell membrane by utilizing an indirect immunofluorescent method to obtain the cell line. The invention not only can be used for the separation of canine distemper viruses and the titration of the viruses, but also markedly shortens the time for separating the viruses, improves the yield of the viruses, and forcefully promotes researches on the molecular characteristics, such as phenotypes, genotypes and the like of the canine distemper viruses.

The invention discloses a method of quickly analyzing group and number of blood lymphocytes of various common crustaceans according to the principles that with the particle structure in the blood lymphocytes of crustaceans, such as litopenaeus vannamei, procambarus clarkia, eriocheir sinensis and the like, as classification reference, the blood lymphocytes, in different groups, can be discriminated and statistic analysis is automatically completed at the same time with a region, where sample points are located, and auxiliary images as evidences by means of a multi-dimensional panorama analyzer having an image auxiliary function. The method is free of dyeing and avoids influence due to manual steps such as normal dyeing, observation under a microscope and the like; by means of automatic signal analysis of a high-sensitivity detector, the method has good repeatability. The multi-dimensional panorama analyzer has high-throughput detection performance, so that the reliability of a result from tens of thousands cell sample points is far higher than that of manual counting. The method is very low in usage amount of reagents and consumables and is low in cost due to trace amount detection. The method not only can be used for scientific analysis and daily monitoring, but also can supply technical support to classifying collection of the blood lymphocytes, so that the method has great application value.

Each 100 ml of blood anticoagulant contains trisodium citrate 3.31-3.32g, citric acid 0.01-0.05g, glucose 2.45-3.37g and the rest is water. The tests show that after the external blood is undergone the process of anticoagulation treatment it can prolong the stroage life of lymphocyte to 48-72 hr. from original 4-6 hr. Said invention also provides its application method.

The invention discloses a peripheral blood lymphocyte micronucleus detection kit and a detection method thereof. The detection kit provided by the invention comprises erythrocyte hemolysin, a red fluorescent dye solution, a lysis solution and a positive standard product, wherein the lysis solution comprises sodium chloride, sodium citrate, Triton X-114, green fluorescent dye and RNase; and a peripheral blood lymphocyte micronucleus detection method based on the kit and a flow cytometry detection method is provided. An experiment proves that the peripheral blood lymphocyte micronucleus detection kit and the peripheral blood lymphocyte micronucleus detection method are quick and accurate; on the premise of guaranteeing objective and true experimental results, the detection cost is reduced, and the experiment has repeatability and can be widely applied to clinical diagnosis and monitoring of occupational exposure genetic damage of healthy people, rehabilitation detection of genetic damage of cancer patient and cellular genetic science research.

The invention provides a method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of an RNA interference technology. The method comprises the following steps: (1) synthesizing dsLvBip with sequence specificity according to a cDNA sequence of a Bip gene of litopenaeus vannamei; and (2) diluting dsLvBip, controlling the injection volume at 50 mu L and injecting dsLvBip into the litopenaeus vannamei according a dosage of 1 mu g / g. The method provided by the invention can be used for simply, conveniently, rapidly, efficiently and specifically inducing blood lymphocyte of litopenaeus vannamei to generate UPR.

The invention provides a continuous culture technology of prawn cells by establishing an effective prawn cell 3D culture and subculture method. The continuous culture technology can be applied to establishment of prawn immortal cell lines. The addition ratio of matrigel is optimized, and thus, a preparation method of the matrigel for 3D culture of the prawn cells is provided. A formula of a complete culture medium of the prawn cells is further optimized, the complete culture medium of the prawn cells is selected as an anticoagulant and a diluent of prawn blood lymphocytes, a 3D culture mode of attached growth on the surface of the matrigel is selected, thus, the separation and 3D culture technology of the prawn peripheral blood lymphocytes is provided, the prawn peripheral blood lymphocytes are attached to the surface of the matrigel for growth by a mode of round single cell and cell clusters / balls, and the survival and growth abilities of the prawn peripheral blood lymphocytes are higher than those of the prawn peripheral blood lymphocytes subjected to 2D culture. The invention also provides a passaging method for culturing the prawn cells in a 3D mode, the passaged prawn blood lymphocytes can be well attached to the surface of the matrigel for growth, and the survival rate of the passaged prawn blood lymphocytes can reach up to 90% or above.

The invention belongs to the technical field of cell mediums, and in particular relates to a human peripheral blood lymphocyte medium and a preparation method thereof. The medium provided by the invention mainly consists of the following components: an RPMI1640 medium, fetal bovine serum, collagen, sodium hyaluronate, glucagon, thyrotropin releasing hormone, total glucosides of paeony, linoleic acid, butanediamine, gluconic acid iron salt hydrate, glutathione and polylysine. The medium provided by the invention can be used for effectively inhibiting microorganisms and viruses, and the medium is good in reagent stability and high in transformation rate of cultured lymphocytes and lymphocyte division index.

The invention relates to a method for inspecting lymphocytic subgroup by a single clone antibody SPA red cell garland way. The method comprises the following procedures: first the red cell garland reagent is solved, then the circumference blood leukomonocytes are separated; leukomonocyte suspension is prepared by a Henke balancing salt solution free of Ca and Mg containing 20% serum of a newly borne calf; meanwhile, the sensitized red cell suspension is made into a sensitized red cell application liquid by using a Henke balancing salt solution free of Ca and Mg containing 20% serum of a newly borne calf; then the leukomonocyte suspension and the sensitized red cell application liquid are mixed by 1:1 V / V into a mixed suspension, which is centrifuged after placing still under 18-28 centigrade, then is placed still for 15-45 min under 4 centigrade; finally the mixed suspension is absorbed 15 or 20 times by a pipet having a sucking head, the absorbed suspension is made into cell smear, which is naturally dried, dyed and counted by a microscope with high magnification. Comparing with prior McAb-A-E method, the invention is of simplified detection process, shorter period, quantitative controlling, and can meet clinic detection.

The invention relates to an indirect method for using a monoclonal antibody SPA red cell rosette to detect lymphocyte subsets. The method firstly prepares a primary antibody sensitized red blood cell preservation solution and a secondary antibody sensitized red blood cell preservation solution; secondly, the peripheral blood lymphocytes are separated, and then a secondary antibody sensitized red blood cell application solution and a primary antibody sensitized lymphocyte suspension are prepared by a calcium-free and magnesium-free Hanks balanced salt solution which contains 20 percent newborn calf serum; then the primary antibody sensitized lymphocyte suspension and the secondary antibody sensitized red blood cell application solution are mixed by the volume ratio of 1: 1 to form a mixed suspension; a centrifugation is carried out after the mixed suspension is statically settled under 18 - 28 DEG C, and then the mixed suspension is statically settled for 15 - 45 minutes at the temperature of 4 DEG C; finally, a pipette which is provided with a suction head is used for blowing and sucking the mixed suspension for 8 - 15 times, then cell smears are prepared and calculated by a high-power lens after natural drying and dyeing. Compared with the original McAb-A-E indirect method, the invention is characterized by simplified inspection process, short period, quantized control and satisfying the requirements of clinical inspection.

The invention belongs to the technical field of immune cell in-vitro culture, and particularly discloses a culture method of umbilical cord blood lymphocyte CIK. The method comprises the following steps: by using umbilical cord blood as a treatment object, separating the umbilical cord blood mononuclear cells, purifying and inducing the CIK cells, culturing and amplifying the CIK cells, and finally, centrifuging to collect the mature umbilical cord blood CIK cells, thereby obtaining the product. The IFN-gamma and alpha-galactose ceramide are added to activate the CIK cells, and CD3 monoclonalantibodies, IL-1 alpha, IL-2 and IL-1 beta are added in the early culture stage to perform continuous stimulation, thereby saving the coating time and enhancing the activation efficiency and amplification efficiency of effector cell groups.

The invention belongs to the technical field of cell culture media, and concretely relates to a human peripheral blood lymphocyte culture medium, which mainly contains the following components and concentration thereof: an RPMI1640 liquid culture medium, Ham's 12, bovine serum, hyaluronic acid 3, casein phosphopeptides, phytagglutinin, lyciumbarbarum polysaccharide, baicalin and nisin. The culture medium provided by the invention can be beneficial for effectively inhibiting actions of microorganisms and viruses, promoting cell proliferation, and shortening cell culture cycle, and has the advantages of high lymphocyte conversion rate and lymphocyte division index, and good reagent stability.

The invention belongs to the field of immunodetection and relates to a mycobacterium bovis infection detection kit meditated by recombined fusion protein and a method thereof. The detection reagent comprises combined fusion protein rE6-M63-H65 used as a specific stimulation origin; the combined fusion protein can effectively stimulate sensitized peripheral blood lymphocyte cultured in vitro to release Gamma interferon (IFN-Gamma) at a high level. The cow IFN-Gamma release test established by using the detection reagent rE6-M63-H65 combined fusion protein as the stimulation origin overcomes the insufficiencies of serology detection method and the IFN-Gamma release test with PPD as the stimulation origin, thus enjoying very high sensitivity and specificity and distinguishing mycobacterium bovis infection from mycobacterium avium or non-pathogenic mycobacteria infection and even distinguishing the mycobacterium bovis infection from BGG immunity; therefore, the detection kit and the method of the invention can be effectively used to detect the mycobacterium bovis infection.

The invention belongs to the field of immunodetection and provides a reagent and a method for detecting bovine mycobacterial infection. The detection reagent comprises a recombinant protein mixture used as a specific stimulation source, wherein the recombinant protein mixture can be used for stimulating animals infected with bovine mycobacteria to generate a DTH (Delayed Type Hypersensitiity) reaction and stimulating peripheral blood lymphocyte to release IFN (Interferon)-gamma. The defects of the prior art are overcome by the detection reagent, and the detection reagent has the advantages of good biosafety, low cost and feasibility in standardized production and can be effectively applied to the clinic detection of the bovine tuberculosis.

The invention discloses a monoclonal antibody against an extracellular domain of a goose CD3 epsilon chain and an application of the monoclonal antibody in detection of goose CD3<+> T lymphocytes. Amplification is performed by adopting goose thymus cDNA as a template to obtain a goose CD3 epsilon gene, according to the characteristics of a GoCD3 epsilon extracellular domain gene, a pair of specific primers are designed, amplification is performed to obtain the goose CD3 epsilon extracellular domain gene (CD3 epsilon ex) and the CD3 epsilon ex is expressed by virtue of escherichia coli; the recombinant plasmid containing the CD3 epsilon ex is adopted as an immunogen to immunize BALB / c mice, and the recombinant protein rGoCD3 epsilon is used for booster immunization to obtain a hybridoma cell strain (3C11) capable of stably secreting the monoclonal antibody against the extracellular domain of the goose CD3 epsilon chain. The monoclonal antibody can react with the purified recombinant protein rGoCD3 epsilon and can have a specific reaction with separated goose peripheral blood lymphocytes. Therefore, a novel and effective technical means is provided for detecting the goose CD3<+> T lymphocytes.

The invention discloses a preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes. The preparation method comprises the steps that peripheral blood lymphocytes are treated by using mitomycin C to obtain the feeder cells, and then the feeder cells and pre-cultured tumor infiltrating lymphocytes are co-cultured. The efficiency of expanding the tumor infiltrating lymphocytes of the feeder cells produced by the preparation method is better than that of the feeder cells produced by gamma-ray irradiation, and meanwhile the cost of using the preparation method is significantly lowered. The preparation method is simple and easy to operate, and can be widely applied and promoted.