A method for early identification of the stability of cho cell lines expressing recombinant antibodies
A technology of recombinant antibody and identification method, which is applied in the field of cell biology, can solve the problems of deviation of results, long time for stability evaluation, etc., and achieve the effect of saving time
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Embodiment 1
[0118] Embodiment 1: the comparison of different apoptosis-inducing reagents processing CHO cell peak pattern
[0119] 1. Take 8 clonal samples of the CHO cell line expressing the recombinant antibody at the 5th generation, count them with Vi-cell (Beckman Company), resuspend the cells in 2ml, and the total number of cells is 2×10 6 , placed in a 12-well plate;
[0120] 2. Add the following apoptosis inducers respectively, mix into the CHO cell line, mix thoroughly, and put in 8% CO 2 Shaker, 170rpm / min, 16h;
[0121] Table 1: Treatment concentrations of different apoptosis-inducing reagents
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[0124] 3. Take out the 12-well plate, transfer to a 96-well deep-well plate, centrifuge at 300g room temperature for 4min, and discard the supernatant;
[0125] 4. Resuspend the cells in 200 μl 1% FBS-PBS in a PCR 96-well plate, centrifuge at 300 g for 4 min at room temperature, and discard the supernatant;
[0126] 5. Resuspend the cells in 200 μl BD Fixati...
Embodiment 2
[0139] Example 2: Comparison of peak patterns after treatment of CHO cells with different fixation and permeabilization methods
[0140] 1. Take 8 clones of CHO cell lines expressing recombinant antibodies (passage times are all 5 generations) for Vi-cell counting, resuspend the cells in 2mL, and the total number of cells is 2×10 6 each, put in a 12-well plate, and make 9 copies;
[0141] 2. Add Actinomycin D (Actinomycin D)-5μM, mix into the cells, mix well, put in 8% CO 2 Shaker, 170rpm / min, overnight incubation for 16h;
[0142] 3. Take out the 12-well plate, transfer to a 96-well deep-well plate, centrifuge at 300g room temperature for 4min, and discard the supernatant;
[0143] 4. Resuspend the cells in 200 μL 1% FBS-PBS in a PCR 96-well plate, centrifuge at 300 g for 4 min at room temperature, and discard the supernatant;
[0144] 5-10. See the table below for processing methods
[0145] Table 3: Different fixed penetration methods
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Embodiment 3
[0158] Example 3: Comparison of Peak Patterns Obtained by Different Fluorescent Secondary Antibodies
[0159] 1. Take 8 clones of the fifth-generation CHO cell line expressing the recombinant antibody for Vi-cell counting, resuspend the cells in 2 mL, and the total number of cells is 2×10 6 , placed in a 12-well plate, in 6 copies;
[0160] 2. Add Okadaic acid (Okadaic acid) 20nM, mix into the cells, mix thoroughly, put in 8% CO 2 Shaker, 170rpm / min, overnight incubation (12-18h.);
[0161] 3. Take out the 12-well plate, transfer to a 96-well deep-well plate, centrifuge at 300g room temperature for 4min, and discard the supernatant;
[0162] 4. Resuspend the cells in 200 μL 1% FBS-PBS in a PCR 96-well plate, centrifuge at 300 g for 4 min at room temperature, and discard the supernatant;
[0163] 5. Add 200 μL of 2% paraformaldehyde to each well, fix the cells, and incubate at room temperature in the dark for 10 minutes (method-3 in Example 2);
[0164] 6. Add 200 μL 1% F...
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