Composition for culturing lymphocytes of laboratory rats and culture method thereof
A technology of lymphocytes and experimental mice, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve problems such as expensive medium, higher requirements for culture technology, and inability to meet cell culture requirements
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Embodiment 1
[0031] Example 1 Acquisition of Spleen Single Lymphocyte
[0032] (1) Clean SD rats with a body weight of about 300g and / or clean BALB / c mice with a body weight of about 25g, CO 2 Killed by cervical dislocation after suffocation, soaked in alcohol solution with a volume fraction of 75%, flipped SD rats and / or BALB / c mice with tweezers during the soaking process, so that the alcohol solution fully contacted the body surface and hair of the mice, 5 After -10 minutes, the SD rats and / or BALB / c mice were taken out and placed in a sterile tray, the abdomen was wiped dry with sterilized cotton balls, and the spleen was aseptically taken out.
[0033] (2) The removed spleens were placed in 6 cm-diameter petri dishes, which contained 2 mL of RPMI-1640 medium pre-cooled to 4°C. Place two 6cm Petri dishes on a wide-mouthed container filled with crushed ice to maintain the environment of the isolated spleen at 4°C. This experiment should be kept in a low temperature environment (recomm...
Embodiment 2
[0038] Example 2 Spleen Lymphocyte Classification
[0039] (1) Get the single lymphocyte obtained, press 10 with 75% ethanol or 4% paraformaldehyde 6 cells / ml to prepare a lymphocyte suspension and fix overnight at 4°C.
[0040] (2) Take the fixed cells, wash them twice with PBS, remove the fixative completely, add FITC-CD3 and PE-CD19 monoclonal antibodies respectively, stain at room temperature for 30 minutes, wash twice with PBS, and remove unbound fluorescent antibodies .
[0041] (3) Lymphocytes after the above staining, in PBS by 10 5 cells / ml to prepare the cell suspension, and analyze the ratio of CD3+ and CD19+ cells by flow cytometry, representing the percentages of T lymphocytes and B lymphocytes, respectively.
[0042] (4) The test results showed that the ratio of T cells and B cells before spleen lymphocyte culture was (T / B): 0.87±0.09%.
Embodiment 3
[0043] Embodiment 3 culture medium optimization
[0044] The spleen lymphocyte culture medium in this embodiment includes RPMI-1640 medium, and concanavalin A, phytohemagglutinin, β-mercaptoethanol, mannitol, sodium azide, fetal bovine serum, sodium pyruvate, L-glutamine Amides, non-essential amino acids, penicillin-streptomycin and dimethyl sulfoxide and other substances, there is no sequence requirement for the addition of various substances. The ratio of different substances in the following table is used to optimize the medium, and the initial number of cultured cells is 5×10 6 / ml. After culturing for 7 days, count the cells according to the counting method mentioned above, classify the cells according to Example 2, and calculate the ratio of T and B cells. The culture results showed that the amplification factor of combination 5 in Table 1 could reach 10.67 times, and the ratio of T / B lymphocytes was 0.93±0.03%, which was the best combination. There was no significant...
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