56 results about "Trypan blue" patented technology

Human non-embryonic adult totipotent and pluripotent stem cells are isolated in a simplified serum-free and feeder cell-free process. Most remarkably, certain stem cells, and especially BLSCs, are extremely small, fail to exclude trypan blue, but are nevertheless able to proliferate from even high dilutions. Therefore, so obtained stem cells can be used to prepare true monoclonal stem cell populations, which are useful in numerous uses, including therapeutic, prophylactic, diagnostic, and research uses.

The invention discloses a full-automatic cell non-staining image recognizing and counting method. The full-automatic cell non-staining image recognizing and counting method comprises the following steps of: selecting a full-automatic cell counting meter through an image recognition method, adding a cell suspension to be detected into a counting sheet directly, and inserting the cell suspension to be detected into a counting meter; lowering the receiving capability of a light sensing chip to the light intensity of a red light R and a green light G, intensifying the receiving capability of the light sensing chip to the light intensity of a blue light B, and regulating the aeration time and gain manually so as to change the background of a display to be light blue, and take round hot spots as images of active cells, and take the round blue spots darker than the background color as images of dead cells; and analyzing through cell image recognition software, thereby obtaining accurate large-small cell activity analysis data. According to the full-automatic cell non-staining image recognizing and counting method, dyes are not required to be used for counting cell activities; latent toxicity brought to cells to be detected by trypan blue is prevented; detected cells can continue to live, and the activities of the cells are counted during the culturing process; and meanwhile, according to the full-automatic cell non-staining image recognizing and counting method provided by the invention, non-staining real-time on-line cell counting can be achieved, and the method can be widely applied to the field of life science research.

The invention discloses a squaliobarbus curriculus fin cell line as well as an establishing method and an application thereof. The preservation number of the squaliobarbus curriculus fin cell line is CCTCC NO: C201642; the squaliobarbus curriculus fin cell line is obtained from the tail fin of squaliobarbus curriculus through primary culture and subculture; and an adopted culture medium is obtained by cultivating through Leibovitz' sl-15. The squaliobarbus curriculus fin cell line has the biological characteristics as follows: cells are fibroblast-like cells, and the cells are cultivated to an optimal state by the Leibovitz' sl-15 culture medium which contains 20% of fetal calf serum by volume in a constant-temperature incubator at 25 degrees. The cells, which are resuscitated under the growth conditions, are dyed with trypan blue, so that a cell survival rate reaches 82.5% or above; and the cells are fibroblast-like cells which are vigorous in growth, and the cells include 48 characteristic chromosomes. In addition, a plasmid pEGFP-N1 is transferred to the cell line for expressing and displaying fluorescence. The cell line disclosed by the invention is used for supporting growth and replication of grass carp hemorrhage viruses; and the cell line is not only applicable to expression of exogenous genes but also suitable for toxicology.

A method of preparing cell samples for viable cell number quantification with a fluorescence digital imaging microscopy system employing digital thresholding technique. The cell sample is stained with a first, fluorescent dye and treated with a second dye that is able to quench the fluorescence of the first dye. The fluorescent dye accumulates in viable cells only and is used to stain the viable cells. The second dye is excluded from viable cells but enters non-viable cells, thereby quenching the background fluorescence in non-viable cells and the medium. Two examples of dye combinations are described: fluorescein diacetate used as the fluorescent dye with eosin Y as the quenching dye; and calcein-AM used as the fluorescent dye with trypan blue as the quenching dye. By reducing the background fluorescence, the dynamic range and accuracy of viable cell number measurements are enhanced. In low viability cultures treated with fluorescein diacetate, background fluorescence completely masked viable cells, but digital thresholding and eosin treatment dramatically reduced background fluorescence, producing a linear response over 4 logs of viable cell density.

A convenient observation method of an orchid mycorrhiza microstructure comprises the following steps: Step 1, preparing a root segment material, a stationary liquid, a dissociation buffer and a staining solution; Step 2, fixing the root segment, and completely immersing the orchid root segment in the stationary liquid; Step 3, slicing the root segment, and shifting the sliced root segment into a clear water-containing container for later use; Step 4, a step of dissociation of the root material: immersing the root material into a KOH solution for dissociation; Step 5, a staining step of the root material: the dissociated root material is placed into a trypan blue solution for staining; and Step 6, carrying out preforming and mounting on the root material, and carrying out microscopic examination with an optical microscope. According to the invention, whether cells in sight are infected by mycorrhiza fungi can be observed clearly, and whether orchid root is infected and the infection degree are detected by statistics of the proportion of the infected cells in sight in total cells in sight. Thus, essential data is provided for the research on coupled growth of orchid.

The invention provides a human primary colon cancer cell separation and culture method. The method comprises the following steps of (1) cutting a fresh colon cancer tissue under an aseptic condition,sealing at 4 DEG C and then carrying back to a lab; (2) disinfecting a tissue processing device; (3) using a pair of operating scissors for separating the colon cancer tissue, placing the tissue intoa precooled sterile PBS liquid containing double antibodies, and washing multiple times to remove blood vessels, fat and necrotic tissues; (4) cutting up the tissues, and using preheated pancreatin and IV type collagenase for digesting respectively; (5) terminating digestion, centrifuging, and removing a supernatant; (6) using trypan-blue for dyeing a cell filter liquor to detect cell activity; (7) after carrying out cell counting, inoculating into a coated culture flask; (8) culturing at 37 DEG C in an environment with 5 percent of CO2; (9) purifying the cells; (10) subculturing the tissues;(11) observing cell morphology; (12) determining cell survival rate; (13) carrying out immunological identification. The invention provides the human primary colon cancer cell separation and culture method which is simple to operate, the number of the obtained cells is large, the survival rate is high, the method is the ideal human primary colon cancer cell primary separation and culture method, and a reliable cell resource is provided for experiments.

The invention provides a test evaluation method for sensitization of a traditional Chinese medicine injection. The method comprises the steps of: treating a traditional Chinese medicine injection by ultrafiltration enrichment with 1-3KD to prepare a sensitized large molecular part; then adding the large molecular part into a peripheral blood mononuclear cell (PBMC) solution; carrying out co-culture for 1-3 days; and employing optimized trypan blue or fluorescent dye to dye living cell numbers, in order to calculate and evaluate sensitization strength of the traditional Chinese medicine injection. The method has advantages of convenience, rapidness and sensitivity, can be widely used in test and evaluation on sensitization and irritation of various traditional Chinese medicine injections, and provides scientific basis for safety evaluation and quality control of traditional Chinese medicine injections, and safety of clinical medication.

The invention discloses a method for removing mouse oocyte nuclei by adopting a zona pellucida solution cavity method. The method comprises the following steps of: performing central slight dripping and peripheral slight dripping in a plastic utensil; making an acid Tyrode's solution react with a zona pellucida under a microscope; forming a pore on the zona pellucida 1-6 seconds later by dissolving till the zona pellucida becomes thin and soft and cytoplasm protrudes outwards; absorbing a first polar body and surrounding cytoplasm out; releasing an oocyte; and denucleating and observing integrality by adopting trypan blue dyeing, wherein the denucleation success rate is over 99.1 percent. In the preparation method, used equipment is simple and the denucleation operation can be completed under a micromanipulator; a reagent has the advantages of simple components, easiness of preparation, low cost, low difficulty of operation, easiness of comprehension, soft denucleation operation, small mechanical damage, quick denucleation and high efficiency; the average denucleation time of each oocyte is 1-6 seconds, the survival rate of each denucleated oocyte is over 97 percent and the success rate of denucleation is over 99 percent; and the method is suitable for researching mouse nucleus transplantation in a Piezo-free laboratory and early events relevant to embryonic development.

The invention discloses a detection method for the adhesion rate and the phagocytosis rate of phagocytes on bacterial. According to the detection method, phagocytes are divided into a control group, a treatment group and a untreated group, the control group cells are treated with trypan blue and cytochalasin D, the treatment group cells are treated with trypan blue or cytochalasin D, the untreated group cells are not treated, bacteria are labeled with CFDA-SE, and after the bacterial and cells interact, the adhesion rate and the phagocytosis rate of the phagocytes are detected through flow cytometry. According to the present invention, the adhesion rate and the phagocytosis rate of the phagocytes on pathogenic bacteria can be rapidly and accurately detected, and the whole experimental process does not exceed 3 h; the required operation steps after the interaction of the pathogenic bacteria and the phagocytes are less, such that the detection of the large batch sample can be performed; the required reagent is less and the price is economical so as to substantially reduce the scientific research cost; and the detection method is suitable for the adherently growing phagocytes and the difficult-detected phagocytes having the suspending growing state, and broaden the research idea to a certain extent.

The invention relates to the technical field of biology, and particularly discloses a method of quickly measuring titer of ascovirus. The method includes: resuspending logarithmic phase Sf9 insect cells; placing into an incubator, standing for culture for 30 min until the cells are attached to walls, abandoning a supernatant culture medium, and replacing with a new Sf-900IISFM culture medium; utilizing a blood ball counting plate to measure cell concentration; taking and well mixing Sf9 insect cell suspension with ascovirus diluent, and inoculating to a 96-hole cell culture tray; disposing in the incubator for culture for 24 h, adding trypan blue dyeing liquid into each hole, and recording dyed cell number and total cell number of each hole; calculating Sf9 insect cell death rate of each hole, determining that the corresponding hole is infected with ascovirus if cell death rate of each hole is lower than that of a control group, determining that the corresponding hole is not infected with ascovirus if not, and calculating TCID50 of ascovirus according to a formula. The method combines cell dyeing, can be used for quick measuring of titer of ascovirus and is more accurate in measuring result.

The present invention provides a bacillus subtilis heterologous protein expression quantity improving method. Recombinant plasmid-contained B.s ubtilis is directly induced for mutation by ARTP and starch-trypan blue panel prescreening, 96 orifice plate high-throughput screening and shaking flask fermentation test to obtain a high enzyme activity mutant strain, and the enzyme activity is increased by 31.4%. The ARTP technology used in the method is a biological mutagenesis technology very wide in application range; during induced mutation, the recombinant plasmid-contained B.s ubtilis strain is directly induced for mutation, the recombinant plasmid conversion step can be eliminated after the induced mutation, efficiency is high; at the same time, and by combination with a 96 orifice plate for high-throughput screening, the efficiency for screening of direct mutation strains can be greatly improved, so that the method can rapidly screen a heterologous protein high yield B.s ubtilis strain, and has important economic value.

The invention discloses a method for rapidly identifying disease resistance by inoculating grape powdery mildew to detached grape leaves. According to the technical scheme, the method comprises the following steps: separating grape powdery mildew strain microspecies, purifying and infecting; performing rDNA sequence amplification and evolution analysis so as to obtain a grape powdery midew NAFU1 with high pathogenicity; analyzing the preservation and the expanding propagation of the strain; inoculating the strain with the detached grape leaves, dyeing by using trypan blue, inoculating to sick leaves in different periods, and observing indexes such as the time of spore germination, growth velocity, hypha length and necrocytosis, thereby rapidly and accurately detecting the disease resistance of grape. The grape powdery mildew preserved and amplified by using the method is high in sporulation quantity, the spore is fresh and high in pathogenicity, the defects that the conventional field identification period is long, the repeatability is poor and the weather influence can be caused are overcome, not only is the efficiency in detecting the disease resistance of a grape germplasm resource in large scale improved, but also direct reference values in rapidly identifying the disease resistance of the grape germplasm resource as well as hybrid progeny and transgenosis plants of the grape germplasm resource in large scale are achieved.

The invention discloses a FISH pretreatment method and pretreatment fluid for an FFPE sample. The pretreatment fluid comprises a pretreatment fluid I, a pretreatment fluid II and a pretreatment fluid III, wherein, the pretreatment fluid I is at least one selected from the group consisting of an EDTA solution, a citric acid buffer and a sodium sulfocyanate solution, the pretreatment fluid II is at least one selected from the group consisting of pontamine sky blue with a concentration of 0.01 to 0.5% and trypan blue with a concentration of 0.025 to 1%, the pretreatment fluid III is sodium borohydride with a concentration of 0.1 to 5%, and the above-mentioned substances are weighed according to a mass volume ratio. The pretreatment method is composed of a plurality of steps. The FISH pretreatment method and pretreatment fluid provided by the invention can substantially reduce FISH autofluorescence and background of liver cancer FFPE.

The invention discloses a detection method of the biological activity of vascular endothelial growth factors. The method comprises the following steps of (1) adding trypan blue into collected human umbilical vein endothelial cells; performing counting after the uniform mixing; regulating the cell concentration to (2 to 10)*10<4>mL according to the counting result; (2) culturing the regulated cells for 3 to 10h in a CO2 culture box; (3) performing concentration gradient dilution on the endothelial growth factors; adding all gradient dilution endothelial growth factor solutions into cells cultured in the step (2); after the uniform mixing, performing culture for 80 to 100h in the CO2 culture box; (4) adding Alma blue color developing liquid into a mixed solution of the vascular endothelial growth factors and the cells cultured in the step (3); after uniform mixing, performing culturing for 1 to 10h in the CO2 culture box; (5) performing fluorescence intensity detection on the sample cultured in the step (4). Through the activity detection on the vascular endothelial growth factors, the use quantity of the vascular endothelial growth factors in different batches is regulated, so that the activity detection result is stable and more accurate.

The invention provides a mouse skeletal muscle satellite cell separation and culture method. The mouse skeletal muscle satellite cell separation and culture method comprises following steps: 1, mice are killed through intraperitoneal injection of pentobarbital sodium, and are immersed in 75% alcohol for disinfection; 2, the mice are fixed on plates with the venter posterior surfaces upward, hind limb skin is peeled under aseptic conditions, and thighbone muscle tissues are collected; 3, in an aseptic operation bench, PBS washing is carried out for three times, surface blood vessels, tendons, and connective tissues are removed; 4, the processed tissues are cut into pieces, and are subjected to digestion with preheated trypsin and IV type collagenase respectively; 5, digestion is stopped, centrifugation is carried out, and an obtained supernatant is removed; 6, a cell filtrate is collected, and trypan-blue drying detection is adopted to detect cell activity; 7, after cell counting, a complete medium is adopted for resuspension inoculation into a coated culture flask for culture at 37 DEG C at 5% CO2 environment; 8, differential attachment method is adopted to remove fibrocytes; 9, cell morphology is carried out; 10, ELISA method detection is carried out; and 11, RT-PCR detection is carried out. The mouse skeletal muscle satellite cell separation and culture method is simple in operation, and is high in survival rate; the number of obtained cells is large; the mouse skeletal muscle satellite cell separation and culture method is an ideal method, and is capable of providing experiments with reliable cell resources.

The invention relates to the technical field of injection reagent, in particular to a submucosal injection solution composition for an endoscope and a preparation method. The composition is prepared from 0.01 w/v% to 0.5 w/v% of hyaluronic acid, 0.005 w/v% to 0.5 w/v% of carboxymethyl chitosan, 0.001 w/v% to 0.1 w/v% of coloring agent and the balance of phosphate buffer solution, wherein the hyaluronic acid is selected from sodium hyaluronate, magnesium hyaluronate, calcium hyaluronate or zinc hyaluronate; the substitution degree of the carboxymethyl chitosan is greater than 0.85, and the dynamic viscosity of the carboxymethyl chitosan is 500-5000 mPa*s; and the coloring agent is selected from trypan blue, methylene blue, indigo blue, gentian violet or indocyanine green. The hyaluronic acid, the carboxymethyl chitosan and the coloring agent are combined for use, relatively long mucous membrane lifting time is kept, the lesion boundary is clearly displayed, postoperative bleeding and infection are reduced, and the submucosal injection solution can serve as an endoscopic submucosal injection solution to be applied to endoscopic surgery.

The invention discloses a system for characterizing macrophage phagocytosis microorganisms of pompanos. The pompanos are dissected and separated through a surgical knife and tweezers to obtain heads and kidneys; the heads and the kidneys are abraded through a cell sieve to obtain cell suspension containing macrophages, and the macrophages of the heads and the kidneys are obtained through a Percoll solution density gradient centrifugal tube. The survival amount of cells cultured in vitro through a trypan blue dyeing manner is counted, and the survival condition in one week is detected. The system is used for carrying out a phagocytosis experiment, and phagocytosis conditions of nocardia, yeast and the like of the pompanos by the head and kidney macrophages of trachinotus ovatus are determined, and a phagocytosis function is observed. An experiment result of the system shows that the macrophages of the pompanos can be extracted very well, and the survival condition and the phagocytosis condition of the macrophages of the pompanos can be observed very well. The system disclosed by the invention lays a foundation for further researches on physiology, pathology and toxicology of the macrophages of the pompanos.

The invention discloses a method for detecting CHO cell proliferation by using optical analysis and application thereof. The method comprises the following steps: (1) converting pEGFP-N2 into Escherichia coli DH5alpha, shaking the bacteria, and extracting endotoxin-free plasmids; (2) inoculating CHO cells into a pore plate, performing transient transfection on the plasmids in the step (1) into the CHO cells by adopting a lipofection transfection method when the cell growth density is 70 percent; (3) screening the transfection cells by adopting geneticin G418, performing resistance screening of over 30 generations at least, thereby obtaining a cell strain which can stably express the GFP; and (4) performing multiplication culture on the cell strain which can stably express the GFP, digesting the cells, performing cell counting by adopting trypan blue, detecting the fluorescent intensity by adopting an enzyme linked immunosorbent assay detector, and judging the CHO cell proliferation conditions based on a linear relation between GFP fluorescent intensity in the cells and the cell number. The method disclosed by the invention has the advantages of operability, detection result accuracy, high repeatability, high sensitivity, non-toxicity and the like.

The invention discloses a method for promoting proliferation of intestinal cancer cells based on CCL20 and CXCL8. The method comprises the following steps: 1, selecting a proper experimental material; 2, preparing a main reagent and storing the prepared reagent; 3, proliferating the cells, and carrying out different experimental tests on the proliferated cells; 4, carrying out trypan blue discharge dyeing experiment on the cells, and calculating the survival rate of the cells; 5, carrying out immunohistochemical staining on the cells. Compared with the prior art, multiple groups of experiments are adopted for comparison, the CCL20 stock solution and the CXCL8 stock solution with different concentrations can be respectively arranged for proliferation culture of the intestinal cancer cells; on one hand, the influence of the chemotactic factor concentration on the proliferation rate of the intestinal cancer cells under the condition of the same chemotactic factor is determined; on the other hand, the influence of a single chemotactic factor and two chemotactic factors on the proliferation rate of the intestinal cancer cells under the same chemotactic factor concentration condition can be determined, and the influence of the chemotactic factor concentration and the chemotactic factor number on the proliferation rate of the intestinal cancer cells can be known.

The invention discloses application of 450-490 nm LED-blue-light combined with arsenic trioxide in osteosarcoma treatment. Trypan-blue dyeing, cell counting and the EdU dyeing method are adopted to detect the influence of the 450-490 nm LED-blue-light combined with the arsenic trioxide on osteosarcoma cell growth and proliferation, scratch tests and Transwell tests are applied to detecting the influence on cell migration and invasion capabilities, and gamma-H2AX immunofluorescent staining is applied to detecting the influence on cell nucleus damage. Test results show that through combination of the 450-490 nm LED-blue-light and As2O3, curative effects of independently used small-dosage As2O3 can be improved, the toxicity is reduced, the growth, proliferation, migration and invasion of osteosarcoma cells can be effectively inhibited, the cell nucleus damage is induced, and then the purposes of relieving and treating osteosarcoma are achieved. The clinical application of the 450-490 nm LED-blue-light combined with arsenic trioxide is simple and low in medical treatment cost, pain of treated patients is reduced at the same time, and a new technological means is provided for the osteosarcoma treatment.

The invention discloses an in-vitro culture method for improving the activity of umbilical cord blood derived NK cells. The method adopts the following raw materials: a biosafety cabinet, a flow cytometer, a horizontal temperature control centrifuge, a sterilization pot, a CO2 incubator, an inverted microscope, a three-classification blood analyzer, a cell counter, a pipettor, refrigerators with the temperatures of -20 DEG C and 4 DEG C, a 75cm<2> culture bottle, a 225cm<2> culture bottle, a 2L culture bag, a 10ml transfer pipette, a 50ml efficient centrifuge tube, 15ml and 50ml centrifuge tubes, a 250ml centrifuge tube, medical sterile gloves, disposable sterile cap and sterile gloves, a serum-free culture medium LonzaX-VIVO15, recombinant interleukin 2, recombinant interleukin 15, a 0.4%trypan blue solution, physiological saline, human lymphocyte separation liquid, gamma interferon and recombinant interleukin 1[alpha]. According to the method, lentinan is added into related cytokines to culture the umbilical cord blood NK cells, so that the cost of culturing the NK cells by the method is not high, the proliferation speed is increased by 13-15 days, the proliferation multiple isincreased by about 270 times, the cell activity is high, and the cell phenotype is 91%.

An ophthalmic composition comprising indigo carmine or indigo carmine and trypan blue for identifying intraocular structures and membranes within the eye, and methods of delivering and using the ophthalmic composition for surgical treatment of the eye, including glaucoma and cataract surgery.