Preparation method for immortalized lymphocytes
A lymphocyte and immortalization technology, which is applied in the field of preparation of immortalized lymphocytes, can solve the problems of cumbersome preparation methods and low success rate, and achieve the effects of wide host range, high success rate and improved sensitivity
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Embodiment 1
[0028] Step 1: Immunizing mice with specific antigens.
[0029] When implementing it:
[0030] Select four-week-old BAL B / C mice to immunize the mice. According to the different characteristics of the antigen, it is divided into the following two schemes:
[0031] (1) Soluble antigen: The immunogenicity is weak, and adjuvants are generally added. For haptens, immunogens should be prepared first, and then adjuvants should be added. Commonly used adjuvants: Freund's complete adjuvant, Freund's incomplete adjuvant.
[0032] Initial immunization: 1-50 μg antigen plus Freund's complete adjuvant subcutaneous multi-point injection or intrasplenic injection (generally 0.8-1ml, 0.2ml / point)
[0033] After 3 weeks, the second immunization: the dose is the same as above, plus Freund’s incomplete adjuvant subcutaneously or ip (intraperitoneal injection) (ip dose should not exceed 0.5ml)
[0034] After 3 weeks, the third immunization: the same dose, no adjuvant, ip (5 to 7 days later, b...
Embodiment 2
[0053] Step 1: Immunizing mice with specific antigens. (step 1 with embodiment 1)
[0054] Step 2: Obtain lymphocytes from the spleen.
[0055] When implementing it:
[0056] (1) Three days after the booster immunization, the mice were killed by dislocation of the neck and disinfected by immersing in 75% ethanol. Take out the spleen of the mouse in the ultra-clean bench, grind the spleen with 4-5ml 1× lymphocyte separation medium (shake well before taking it) in a sterile grinding tube, and filter it with a 200-mesh sieve after grinding to ensure that the isolated cells for a single cell suspension.
[0057] (2) Immediately transfer the separation liquid containing the spleen cells to a 15ml centrifuge tube, cover with 200-500 μL of 1640 medium, and centrifuge at 800 g for 30 min at room temperature.
[0058] (3) Aspirate the lymphocyte layer, add 10ml of 1640 medium, and wash upside down. Cells were collected by centrifugation at 250 g for 10 min at room temperature. Po...
Embodiment 3
[0065] Step 1: select mice naturally infected with leukemia virus, and take spleen under aseptic conditions.
[0066] Step 2: obtaining lymphocytes from the spleen, culturing the lymphocytes, and obtaining immortalized lymphocytes.
[0067] When implementing it:
[0068] (1) Grind the spleen with 4-5ml 1× lymphocyte separation medium (shake well before use) in a sterile grinding tube, and filter it with a 200-mesh sieve after grinding to ensure that the isolated cells are a single cell suspension.
[0069] (2) Immediately transfer the separation liquid containing the spleen cells to a 15ml centrifuge tube, cover with 200-500 μL of 1640 medium, and centrifuge at 800 g for 30 min at room temperature.
[0070] (3) Aspirate the lymphocyte layer, add 10ml of 1640 medium, and wash upside down. Cells were collected by centrifugation at 250 g for 10 min at room temperature. Pour off the supernatant, resuspend the cells with lymphocyte culture medium, and culture them with soft agar...
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