118 results about "Cell Pellet" patented technology

A method for selectively growing epithelial cells or carcinoma cells in vitro without fibroblast overgrowth comprises (a) suspending a cell pellet comprising digested epithelial or carcinoma cells in a first growth medium, the medium comprising D-valine MEM, methyl cellulose, fetal serum, glutamine and an antibiotic; wherein the methyl cellulose is present in the medium at a concentration sufficient to inhibit growth of fibroblast cells present in the cell pellet; (b) adding the suspension to a cell culture vessel comprising an inner surface which has been at least partially coated with an attachment medium comprising a protein extract, D-valine MEM, glutamine and an antibiotic; and (c) incubating the suspension in the coated vessel to allow selective growth of the epithelial cells or carcinoma cells.

The invention discloses an FFPE reference product for gene detection and a preparation method and application of the FFPE reference product. The preparation method comprises the following steps that S1, cell culture is performed on a tumor cell line, and cell pellets are collected; S2, formalin fixation is performed on the cell pellets, then sepharose gel wraps the cell pellets to form cell aggregates, and the cell aggregate are prepared into cell paraffin blocks; S3, genomic DNA of the tumor cell line in the cell paraffin blocks is extracted, and genetic mutation frequency determination is performed on the genomic DNA of the tumor cell line; S4, the genomic DNA of the tumor cell line containing a target mutation site is mixed into a DNA mixture of a target mutation frequency, and the DNAmixture is the FFPE reference product for gene detection. According to the technical scheme, formalin fixation and paraffin wrapping are performed on the cells cultured by tumor cell line, so that thesituation of clinical samples can be better simulated.

The invention relates to a method for obtaining a large number of adipose-derived mesenchymal stem cells from fat. On one hand, the invention relates to a method for isolating and culturing the adipose-derived mesenchymal stem cells. The method comprises the steps that fat samples doped with swelling fluid and obtained by liposuction are placed in a centrifuge tube, and centrifugation is conducted; the uppermost layer of fat is removed, and suspended fat particles and underlying cells are transferred and precipitated respectively for later use; mixed liquid of digestive enzyme is added in theobtained fat particles, shaking digestion is conducted, the digestion is terminated, and resuspension is conducted on cell precipitate; red blood cell lysis buffer is added to underlying cell precipitate, the underlying cell precipitate is allowed to stand, and splitting decomposition, centrifugation and resuspension of the cell precipitate are conducted; cell suspension is combined, and adipose-derived mesenchymal stem cells which serve as primary dipose-derived mesenchymal stem cells are isolated; culturing and reproduction are further conducted on the primary dipose-derived mesenchymal stemcells. The invention further relates to a adipose-derived mesenchymal stem cell preparation prepared by the method, the mixed liquid of the digestive enzyme for isolating the adipose-derived mesenchymal stem cells, and a culture medium for culturing the adipose-derived mesenchymal stem cells. Excellent technical effects as described in a description are shown in all aspects of the method for obtaining a large number of adipose-derived mesenchymal stem cells from the fat.

A method for promoting a differentiation of stem cells into insulin producing cells is provided. The method includes steps of suspending the stem cells in a first culture medium, aggregating the stem cells to form a cell pellet, and culturing the cell pellet in a second culture medium to promote the differentiation of the stem cells of the cell pellet into the insulin producing cells.

The invention discloses a method for separating fetal nucleated red blood cells in maternal peripheral blood. The method comprises the following steps: (a) enriching nucleated cells, namely, (1) collecting whole blood, (2) centrifuging, and collecting cell precipitates, (3) preparing cell suspension, namely adding a cell culture medium to the cell precipitates, and uniformly mixing so as to prepare the dilute cell suspension, (4) putting a cell separating medium in a centrifugal tube in advance, and adding the dilute cell suspension to a layering liquid surface, (5) centrifuging so as to obtain a cell layer, and (6) sucking up the cell layer, adding Hank, s liquid, uniformly mixing, centrifuging at a room temperature, and washing cells, and (7) resuspending the cells; and (b) screening the fetal nucleated cells in the maternal blood, to be specific, (9) separating out the fetal nucleated red cells, and (10) carrying out quadruple morphology identification on nucleated red cell pictures obtained by sorting, and collecting the nucleated red cells meeting the standards for extracting the fetal whole genome DNA (deoxyribonucleic acid). According to the method, the purity and the recovery rate of the fetal nucleated red blood cells obtained by the separation and purification of the maternal peripheral blood are high.

The invention discloses a method for establishing high-efficiency somatic embryogenesis regeneration plants of plantain and braziliana. In this method, before the induction of somatic embryos with suspension cells, they are pre-cultured with M2 liquid medium without 2,4-D for 7 to 10 days, and the cell clusters with a relatively consistent size and growth state are screened out through a sieve, and inoculated into somatic embryos. on the induction medium. Pre-cultivation can increase the induction rate of somatic embryos and the subsequent plant transformation rate by about 1 times; the cell clusters with a size ranging from 154 μm to 900 μm obtained through sieves have the best induction effect of somatic embryos; The optimum inoculum volumes were 0.4ml 20% SCV and 1ml 20% SCV respectively. In addition, adding ABA and ascorbic acid on the prior art somatic embryo induction medium of bananas at the initial stage of somatic embryo induction prevents to a certain extent the phenomenon that plantains are prone to browning during somatic embryo induction, and can effectively improve plantain and Frequency of somatic embryogenesis in embryogenic suspension cells of Banana brasiliensis.

The invention provides a universal cell processing kit and an application method thereof, and relates to a kit for separating stem cells from human cord blood, bone marrow and peripheral blood and an application method of the kit. The kit contains a reagent A (cell diluent), a reagent B (high-molecular-weight cell precipitant) and a reagent C (medium-density layering agent). The application method of the kit comprises the steps of: firstly, uniformly mixing the cord blood, bone marrow or peripheral blood with the cell diluent, adding the high-molecular-weight cell precipitant, standing for 30 minutes, collecting the supernatant, centrifuging, collecting the concentrated solution, adding the medium-density layering agent to the concentrated solution, centrifuging for 20 minutes, collecting a karyocyte layer and washing with saline for injection 3 times. In the invention, a cell precipitant with a molecular weight of 450000-700000 is used, the survival rate of the separated karyocytes is not lower than 98%, the collection rate of karyocytes is not lower than 85%, and the method provided by the invention is simple, safe and practical, has low cost, can realize industrialized production, and is easy to popularize.

The invention discloses a method for separating adipose-derived stem cells from fat and a preparation in use. On one hand, the method for separating the adipose-derived stem cells from fat comprises the steps of conducting liposuction to obtain a fat sample doped with inflation liquid to be subjected to centrifugation; removing the uppermost-layer fat, and removing suspension fat particles and bottom-layer cell sediment respectively; adding the fat particles into digestive enzyme mixed liquid, conducting vibration digestion, stopping the digestion, conducting centrifugation, and re-suspendingcell sediment; re-suspending the cell sediment at the bottom layer, adding erythrocyte pyrolysis liquid to conduct pyrolysis, conducting centrifugal, and re-suspending the cell sediment; merging to obtain cell suspension, filtering, and separating out the adipose-derived stem cells serving as primary passage. The invention further relates to a method for separating and cultivating the adipose-derived stem cells. The prepared adipose-derived stem cell preparations are used for separating the digestive enzyme mixed liquid of the adipose-derived stem cells, and are used for cultivating a culturemedium of the adipose-derived stem cells. The method for separating the adipose-derived stem cells from fat has various advantages as shown in the description.

A novel suspension hybridization assay was used to determine nucleic acid copy number by flow cytometry. The assay was validated with low copy (lc) products ranging in length from 100 to 2304 bp conjugated to spectrally-distinct polystyrene microspheres. In the example provided herein, these conjugated microspheres were used as multiplex hybridization probes to detect homologous sequences in genomic DNA extracted from cytogenetic cell pellets and labeled with biotin-dUTP. Hybridization was detected with phycoerythrin-labeled streptavidin and analyzed by flow cytometry. Copy number differences were distinguishable by comparing the mean fluorescence intensities of test probes with a diploid reference probe in genomic DNA of patient samples and abnormal cell lines. The assay is capable of distinguishing a single allele and three alleles at a test locus from a biallelic reference sequence, regardless of chromosomal context. The assay is an improvement on previous methods which require prior amplification of locus-specific target DNA because, lc probes provide adequate specificity and sensitivity for accurate copy number determination of homologous targets. Because of its high sensitivity and accuracy, the assay is useful for determination of nucleic acid copy number for a variety of applications, including determination of genomic copy number in humans, animal models of disease and in solution, measurement of transcript levels, forensic DNA analysis, and quality control analysis in agriculture.

The invention discloses a method for utilizing a malignant pleural effusion for separately culturing primary cancer cells. The method comprises the steps of putting a pleural effusion sample into a sample collecting liquid, and centrifuging to remove a supernatant; adding a red blood cell lysis buffer for removing a red blood cell, adding a PBS buffer solution for washing the cells, and centrifuging to remove a supernatant; using a KL culture medium for resuspending a cell precipitate, inoculating in a culture flask, and culturing in a culture box. When the cells are proliferated to more than85 percent of abundance, the cells are washed through the PBS buffer solution, pancreatin-EDTA is digested, and a stop buffer neutralizes digestion reaction; the cells are centrifugally collected, areresuspended through the KL culture medium, and inoculated in the cell flask according to the proportion so as to be subcultured. The primary cancer cells of the pleural effusion obtained by separatedculture through the method provided by the invention can be applied to related research of physiology, pharmacy, new drug research and development and the like, exploration of pathogenesis of relateddiseases of a lung cancer, detection of drug sensitivity of different drugs, screening of anti-cancer drugs, new drug research and development, and establishment of a personalized treatment program aiming at a patient.

An isolated and purified glycoprotein and functional analogues thereof are disclosed. The glycoproteins are characterized by being expressed on at least primitive hematopoietic cells, and being a ligand for L-selectin. The binding activity of the ligand of the present invention to L-selectin is not sulfation-dependent and it is neither inhibited by anti-CD34 antibodies nor by MECA-79 monoclonal antibody and the ligand is resistant to O-sialoglycoprotein endopeptidase activity. Further, the present invention provides a method of performing an overlay adherence assay by using isolated cells or cell lines as a substrate. The cells are prepared as the substrate for the assay using a cytocentrifuge with a modified sample chamber allowing placement of the cytocentrifuged cell pellet to any selected location on the slide as required by the shear conditions employed for any given assay.

The invention discloses a chronic glaucoma animal model establishing method. The method comprises the following steps: (1) collecting conjunctiva tissues of GFP-SD rats, sterilizing, digesting, separating a conjunctiva epithelial layer, and obtaining a conjunctiva matrix tissue block; (2) digesting the conjunctiva matrix tissue block in pancreatin, terminating the digestion by using a cell culturesolution, blowing and beating to form a single-cell suspension, filtering by virtue of a cell sieve, centrifuging, and removing supernatant, thus obtaining conjunctiva matrix cell precipitates; (3) adding an appropriate amount of cell culture solution, blowing and beating to form single cells, counting, performing the in-vitro culture, and obtaining a remarkable amount of conjunctiva matrix cells; (4) digesting the conjunctiva matrix cells by using pancreatin, processing by utilizing the method of the step (2), obtaining the conjunctiva matrix cell precipitate, adding an appropriate amount ofcell culture solution, and obtaining cell suspension; and (5) injecting 10 micro liter of cell suspension by utilizing a micro injector into the anterior chamber of the right eye of a rat, and sufficiently and uniformly mixing. A glaucoma animal model obtained by using the method has the advantages of long-term stability, simplicity, ease, good repetition, and low cost.

The present invention relates to a method for clarification of, and removal of host cell proteins from, a cell broth consisting essentially of viable cells, a culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by contacting the cell broth with a particulate anion exchanger, allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, separating the resulting cell pellet from the supernatant layer. The present invention further relates to a method for the recovery of a secreted desired biological substance from the cell broth by extracting the secreted desired biological substance from the supernatant layer.

The invention discloses a mmethod for obtaining analogous retinal tissues rich in cone and rod cells by using human induced pluripotent stem cells. The method comprises the following steps: digestinghiPSCs to obtain cell pellets, and then carrying out suspension cultivation on the cell pellets to obtain an embryoid body; reinoculating the embryoid body into a culture dish enveloped by Matrigel, and carrying out induced differentiation inside an induced cultivation solution to obtain nerve retina (NR) and RPE; stirring up the NR and the RPE, carrying out suspension cultivation to obtain 3D analogous retina comprising NR tissues, then carrying out continuous suspension cultivation inside an optimized culture solution without retinoic acid, carrying out NR differentiation on all retinal cells, comprising highly matured photoreceptor cells, Rhodopsin positive rod cells, L / M OPSIN positive red and green cone cells and S-OPSIN positive blue cone cells, and thus particularly obtaining the analogous retinal tissues rich in red and green cone and rod cells.

The invention discloses an isolated culture method of rumen epithelial cells of an adult yak. The method comprises the following steps of: collecting rumen epithelial tissues of the adult yak, cleaning the tissues with 70% absolute ethyl alcohol, and digesting the tissues step by combining I-type collagenase and trypsin; filtering and collecting cell filtrate through a nylon filter screen, and adding an MEM culture solution containing 10% fetal calf serum to terminate digestion; cleaning the collected cell precipitate; resuspending the cells by using a low-sugar DMEM complete medium, and theninoculating the cells into a culture flask for culture; and removing fibroblasts by a differential adherent method and a phase difference digestion method to obtain the rumen epithelial cells of the adult yak. According to the method provided by the invention, the rumen epithelial cells of the adult yak can be successfully separated, and the cells which are fast in adherent growth, relatively highin activity and stable in passage are obtained. The method can provide a test cell model for physiological regulation and nutrition absorption mechanism of the rumen epithelial cells of the yak.

The invention provides a simple extraction method of high-purity mouse skeletal muscle satellite cells, which is characterized by comprising the concrete steps that: step 1, bupivacaine hydrochloride is injected at the limb muscle of a mouse; step 2, the mouse is killed, the limb muscle is winkled, vessels, fat and fascias are removed, the muscle is sheared into small granules being 0.5 to 1.5mm<3>; step 3, mixed digestive enzyme is added for incubation, the cell suspension liquid is filtered, the obtained filter liquid is centrifuged, the supernate is abandoned, and a growth culture medium for cell precipitation is suspended again; step 4, 60 percent percoll separation liquid, 20 percent percoll separation liquid and cell suspension liquid are respectively injected along the tube wall, a long-head suction tube is used for sucking the cell suspension liquid at the interface of the 20 percent percoll separation liquid and the 60 percent percoll separation liquid, and cell precipitates are obtained through centrifugation; and step 5, the cell precipitates are cultured, and the mouse skeletal muscle satellite cells are obtained. The method is simple, easy and fast, the purification rate and the yield of the skeletal muscle satellite cells are high, higher multiplication and differentiation capability is realized, and the external gene modification or the internal transplantation can be carried out.

The invention relates to an isolated culture method for chicken enterocyte. The method comprises the following steps: step 1, digesting the intestinal tissue block of a chicken embryo with protease so as to obtain a digestion product; step 2, filtering the digestion product with a cell strainer and collecting cell aggregate with a size of 100 to 500 meshes; and step 3, subjecting the cell aggregate to centrifugation at a speed of 800 to 1500 r / min for 5 to 15 min and collecting cell sediment. The invention further provides the chicken enterocyte obtained by using the method. The method provided by the invention can substantially shorten testing time, save reagents and improve the purity of the isolated enterocyte.

The invention discloses a method for high-throughput three-dimensional cell pellet culture and in-situ drug sensitivity test, and belongs to the field of biotechnology. The method is as below: first,forming an array of droplets on a super-hydrophobic micro-pit array chip; second, forming a three-dimensional cell pellet array on a super-hydrophobic micro-pit array chip; third, adding algin into the super-hydrophobic micro-pit array chip, and fixing the cell pellets in the micro-pit; and fourth, conducting in-situ drug sensitivity test on the super-hydrophobic micro-pit array chip. The method can realize the simultaneous loading of each micro-pit of the chip by using a self-built humidification and aligning device, simply and efficiently realizes the construction of the high-throughput three-dimensional cell pellet micro-array by using hanging drop culture and fluid infusion operation, establishes a method for detecting cell activity on the chip, and utilizes a self-established on-chipcell activity detection method to realize the on-chip drug susceptibility test, thereby avoiding the transfer operation of the cell pellets and the resulting loss and damage of the cell pellets.

The invention discloses a method for preparing Leuciscus sp. chromosomes, and relates to a method for preparing fish chromosomes. The invention aims to solve the technical problems that the blood cells culturing method is difficult to collect blood, long in cells culturing process, high in culturing condition and huge in culturing difficulty for undersized fishes. The method comprises the following steps: 1) injecting PHA and colchicine, preparing a cell suspension, centrifuging, adding a hypotonic solution and centrifuging, and discarding supernatant to obtain a cell pellet; 2) fixing; 3) adding 1 to 1.2ml Carnoy's fixative to the centrifuged cell pellet, uniformly mixing to prepare a cell suspension, sucking the cell suspension into a rubber head dropper, adding 2 to 3 drop of the cell suspension under conditions that the rubber head dropper is 25 to 30cm away from a frozen slide and the angle between the rubber head dropper and the frozen slide is 35 to 50 degrees, uniformly bakingthe frozen slide over flames of an alcohol lamp for 3 to 5 times, and drying naturally at room temperature to obtain the Leuciscus sp. chromosomes. The method according to the invention is short in cells culturing time and easy to culture. The Leuciscus sp. chromosomes obtained by the method according to the invention have more spitting phases and have a good effect.

The invention relates to the field of bioengineering, in particular to a method for harmlessly extracting Chinese sturgeon DNA. The method includes the steps of obtaining a sample, processing the sample, conducting cell lysis, precipitating proteins, precipitating DNA, washing DNA and obtaining DNA. The complete method for extracting Chinese sturgeon mucus is established, Chinese sturgeon DNA can be rapidly and effectively extracted, extracted DNA stripes are clear and free of trailing, and it is shown that extracted DNA is high in quality and free of protein contamination and stable and reliable in result.

The present invention discloses a clinical-level purification, separation, culture amplification and cryopreservation method of fat-derived mesenchymal stem cells. The method comprises the following steps: S1. collecting adipose tissue; S2. Adding a washing liquid to wash the adipose tissue; S3. adding a type I collagenase digestion solution to a culture flask to obtain a fat SVF cell filtrate; S4. centrifuging the adipose SVF cell filtrate to obtain a precipitate, namely, fat SVF; S5. discarding a supernatant to obtain a purified fat SVF precipitate; S6. continuing subculture of the culture flask; S7. performing primary culture to obtain primary cells; S8. digesting and harvesting the primary cells, adding the culture medium and performing resuspension, sampling and counting; S9. centrifuging the cell suspension obtained by S8 again, discarding the supernatant, and culturing the culture flask; S10. when the cell fusion in S9 reaches 80% or more, repeating the S8 and S9, and carrying out digestion and washing to obtain a cell precipitate; S11. Sampling the cell precipitate in S10 for detection; and S12. adding a complete culture solution to the cell precipitate obtained after resuspension in the S11, and after the test result is qualified, transferring the cell precipitate to a cell bank for long-term storage.

The invention provides an adherent culture method of neural stem cells. The method includes the steps of conducting primary culture on forebrain histocytes to obtain a neural stem cell sphere suspension; digesting neural stem cell spheres into suspension neural stem cells, inoculating the suspension neural stem cells into a petri dish wrapped by human fibronectin for culture, and conducting continuous cell culture after the cells grow to a certain integration degree; using digestive enzymes to digest the cells during follow-up continuous cell culture, after the cells shrink, blowing down the cells and then conducting centrifugation to obtain cell sediments for continuous cell culture. The adherent culture method of the neural stem cells has the advantages that monolayer adherent culture of the cells is achieved in a limited culture area, the cell harvest yield per unit area is clear, and the cell quantity after harvest can be ensured; moreover, the cells after the adherent culture are easy to digest, damage to the cells is reduced, and the viability of the obtained cells is high; the used human fibronectin for facilitating the adherent culture of the neural stem cells is low in price and capable of being utilized over and over again, other reagents are all common, and the cost of the unit cell quantity is low.

The invention provides a method for separate-culturing cardiomyocytes. The method includes the following steps that (1) heart tissue is taken, and PBS liquid is used to wash away residual blood; the heart tissue is cut into tissue blocks of 0.1-1mm<3> with ophthalmic scissors, 0.5-5ml of collagenase I is added, and shaking digesting is carried out at 36-37.5 DEG C for 0.5-3 hours; after digestion,2.5-50mL of PBS is added for dilution and stop, centrifuging is carried out at 1200rpm / min for 5 min, and a supernatant is discarded to obtain a cell pellet; (2) the cell pellet is resuspended with 0.5-5mL pancreatin, an elbow pipette is used for blowing-beating for 10-300 times, after digestion fluid is turbid, a culture medium is added to stop pancreatin digestion, and centrifuging is carried out at 1200rpm / min for 5 min, and a supernatant is discarded to obtain primary cardiomyocytes; and (3) after the primary cardiomyocytes is resuspended in the culture medium, the primary cardiomyocytesare inoculated into the culture medium according to 0.1-10x10<5> cells / cm<2>, and the obtained mixture is placed in a culture incubator of 37 DEG C and 5%CO2 for culturing to obtain the cardiomyocytes.

Compositions comprising unprocessed cell pellets of a cellulosome-producing microorganism grown on cellulosic biomass are provided. Further provided are methods for producing the compositions and uses thereof in hydrolysis of cellulosic substrates. In particular, the compositions advantageously contain extracellular beta-glucosidase, either expressed on the cells themselves or extrinsically added to the cell pellets.

The invention discloses a high-throughput verification method of a plant tissue single cell transcriptome sequencing result, and relates to the field of gene sequencing. The method comprises the following steps: S1, degrading plant tissues by cellulose enzymatic hydrolysate to prepare a cell suspension; S2, measuring cell concentration and cell activity in the cell suspension; S3, separating different subcell populations in the cell suspension; S4, degrading the same subcell population by the cellulose enzymatic hydrolysate; S5, performing SMART-seq library building and sequencing on a cell precipitation sample; and S6, taking the cDNA obtained by library building as a template, and detecting the expression quantity of a target gene by adopting fluorescent quantitative PCR so as to carry out verification. Cell walls are degraded twice in sequence by adopting cellulose enzymatic hydrolysate so as to separate cells of different subcellular populations, fluorescent quantitative PCR detection is performed after SMART-seq database establishment, high-throughput verification of gene expression quantity is performed on single-cell and subcellular levels, the test period is short, the test cost is reduced, and the verification operation is simple, convenient, efficient and rapid.

The invention discloses a human normal renal tubule primary cell and in-vitro isolated culture and application thereof. The cell is named human normal renal tubule primary cell XHRN-01, and the preservation number of the cell is CCTCC NO:C201970. An isolated culture method of the primary cell comprises the steps that the clinical postoperative excised tissue near the kidney cancer is acquired in vitro, no cancer cell infection is identified through the pathology, the blood vessels and adipose tissue in the tissue are removed, a tissue sample is subjected to collagenase / trypsin digestion, filtration and low-speed centrifugation to obtain cell pellets, after a red blood cell lysis buffer lyses red blood cells, a COMR complete medium is used for resuspending the cells, and a cell line is constructed through continuous cell culture. The cell can be applied in the physiological study on human normal cells, study on drug toxicity of in-vitro normal cells, study on the pathogenesis of the kidney cancer and related diseases including the renal cancer, chronic nephritis and infectious diseases and study on screening of therapeutic drugs.

The invention provides a preparation method for thyroid and mammary fine needle aspiration cell tissue blocks. The method includes the following steps: a, performing centrifugation on hydrothorax or ascites in advance, and obtaining hydrothorax or ascites supernatant; b, taking the hydrothorax or ascites supernatant to inject into a centrifuge tube; c, injecting a fine needle punctured thyroid andmammary specimen into the centrifuge tube, and then immediately taking 95% of ethanol to inject into the centrifuge tube; and d, performing centrifugation on the centrifuge tube and abandoning the supernatant so that cell sediment can be obtained, adding 4% of neutral buffer formaldehyde fixative, and adopting a paraffin tissue specimen processing program to perform conventional embedding after the cell sediment in the centrifuge tube is solidified. The preparation method can guarantee that tissue fragments and free-floating single cells are not lost; and cytoplasm is good in form and structure preservation, can be sliced continuously or for many times, and is suitable for a plurality of dyeing, and therefore, diagnosis efficacy can be enhanced.

The invention discloses a PBMC cell cryopreservation method, which comprises: (1) centrifuging PBMC cells to obtain a cell precipitation, performing resuspending, adding a 2X cryopreservation liquid drop by drop to maintain the density of a cell suspension at 2*10<7> / ml, and performing split charging to cryopreservation tubes; and (2) putting a program cooler, gradually reducing the temperature to-90 DEG C, and transferring the obtained product to liquid nitrogen for long-term preservation. The invention further discloses a PBMC cell thawing method, which comprises: (1) taking out the cryopreservation tubes with PBMC cells preserved, quickly putting the obtained cryopreservation tubes in a water bath, performing repeated vibration for 5-15 times, and controlling the vibration time in 3 min; and (2) washing the obtained cell suspension with a X-VIVO serum-free medium to maintain the cell density at 1-2*10<6> / ml. According to the cryopreservation method, the cooling rate can be accurately controlled via the program cooler, so that ice crystals are prevented from generation, the cell damage is reduced, and the cryopreservation-thawing rate of PBMC cells after cryopreservation-thawinghas been carried out for 24 h is obviously improved.

A Method for preparing an ATP in vitro phosphorylation sample comprises the following steps of culturing cells; collecting the cells to obtain cellular precipitation; extracting cell protein: placingthe cellular precipitation collected in the step S2 in ice and adding a cell lysis buffer for cell ultrasonic pyrolysis, and performing low-temperature centrifugation after ice incubation to obtain aprotein extract, wherein the cell ultrasonic pyrolysis lasts 10min; adding ATP to the protein extract described in the step S3, making the final concentration of ATP as 5mM, performing constant temperature water bath for 1h at 30 DEG C after uniform mixing to obtain the protein extract; performing protein denaturation after protein concentration measurement; and diluting the sample for storage at-80 DEG C. The invention does not need to stimulate cells with numerous drugs to obtain one phosphorylated protein after another. In this system, we can achieve the phosphorylation of almost all proteins, which plays a very good role in the verification of phosphorylated antibodies.