Method for detecting activity of recombinant human interleukin 12 (rhIL-12) protein
An interleukin and cell technology, applied in the field of detection of recombinant human interleukin-12 protein activity, can solve the problems of experimental stability, repeatability, sensitivity and other difficulties, and achieve broad application prospects, unlimited number of passages, The effect of stabilizing costs
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[0032] Preparation of NKG cells:
[0033] 1. Peripheral blood sample: Take 10ml of peripheral blood from a patient with non-Hodgkin's lymphoma, anticoagulate with heparin, and obtain the consent of the patient.
[0034] 2. Separation of peripheral blood mononuclear cells: After diluting the anticoagulated peripheral blood specimen with 10ml sterile PBS solution, carry out density gradient centrifugation with lymphocyte separation medium, 2200 rpm, 30 minutes, 20 degrees; For mononuclear cells, wash 3 times with PBS solution, 1500 rpm, 10 minutes, 20 degrees; use 250 μl sterile PBS solution to resuspend cells, count, and the cell concentration is 1.1×10 7 / 250μl, store at 4 degrees, and set aside.
[0035]3. Separation and purification of NK cells: Add 20 μl of non-specific mouse IgG to the above cell solution at 4 degrees for 30 minutes to block non-specific binding. Add 100 μl CD56 MicroBeads to the cell solution, shake and mix, 4 degrees, 30 minutes, and mix once every 10 ...
Embodiment 1
[0041] Embodiment 1, detect the activity of recombinant human interleukin-12 protein
[0042] 1. Cell activation and preparation
[0043] Inoculate NKG cells (human natural killer cells) CGMCC No.2901 in cell culture medium I at 37°C, 5% CO 2 Cultured under certain conditions until the logarithmic growth phase; during this period, the cells can be subcultured depending on the growth of the cells.
[0044] The cell culture medium I is composed of α-MEM medium, fetal bovine serum, horse serum and recombinant human interleukin-2, and the ratio of α-MEM medium, fetal bovine serum, horse serum and recombinant human interleukin-2 For: 8ml α-MEM medium: 1ml fetal bovine serum: 1ml horse serum: 1000IU recombinant human interleukin-2.
[0045] Get the NKG cells in the logarithmic growth phase in a 50ml sterile centrifuge tube, 800rpm, 10min, centrifuge, discard the supernatant, wash twice with PBS solution, suspend in 1640 complete medium (1640 culture medium containing 10% fetal bov...
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