Subcloned cell strain TF-1-A2, preparation method thereof and application
A technology of TF-1-A2 and TF-1, which is applied in the field of determination of the biological activity of TF-1 subclone stable cell line TF-1-A2 and NGF products, which can solve the problem of difficult to ensure the repeatability and stability of the results , Large deviation of results, low signal-to-noise ratio, etc., to achieve the effect of easy statistical analysis of specific activity, convenient operation, and high signal-to-noise ratio
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Embodiment 1
[0039] Example 1: Domestication of GM-CSF-dependent TF-1 cells
[0040] 1. Routine culture of GM-CSF-dependent TF-1 cells
[0041] GM-CSF-dependent TF-1 cells were purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, maintained in RPMI 1640 complete medium containing 10% fetal bovine serum, and added rhGM-CSF at a final concentration of 8ng / ml nourish. The cell concentration was maintained at 3 × 10 4 -5×10 5 cells / ml, at 37°C, 5% CO 2 Under the conditions of cultivation, the medium was changed once every 2-3 days.
[0042] 2. GM-CSF-dependent domestication of TF-1 cells
[0043] In the presence of a certain concentration of NGF, the GM-CSF-dependent TF-1 cells were domesticated into NGF-dependent TF-1 cells by gradually reducing the concentration of GM-CSF by serial dilution and serial passage. The specific domestication method is as follows: with the RPMI 1640 complete medium containing 10% fetal ...
Embodiment 2
[0055] Example 2: Cloning of NGF-dependent TF-1 cells
[0056] Use methylcellulose semi-solid medium to clone the domesticated mNGF-dependent TF-1 cells, the specific method is as follows:
[0057] 1) The methylcellulose semi-solid medium used is METHOCULT TM H4100 (Stem cell company, Catalog #04100) methylcellulose concentrate (2.6% concentration, stored at -20°C for future use), fully melted at 4°C before use. Add RPMI 1640 complete culture solution containing 10% fetal bovine serum and mNGF and mix it thoroughly for 2.5-fold dilution, so that the final concentration of mNGF is 25ng / ml, and the final concentration of methylcellulose is 1.0%;
[0058] 2) Take mNGF-dependent TF-1 cells in the logarithmic growth phase, collect the cells by centrifugation at 1000 rpm for 10 min, resuspend in RPMI 1640 complete culture medium containing 10% fetal bovine serum, and adjust the cell concentration to 2×10 4 cells / ml of cell suspension;
[0059]3) Take 1ml of diluted cell suspensi...
Embodiment 3
[0063] Example 3: Screening of NGF-dependent TF-1 monoclonal strains
[0064] According to the method described in Example 1, the dose-effect-response curves of the 15 monoclonal cell strains obtained by the cloning of the methylcellulose semi-solid medium were plotted respectively, and the maximum proliferation effect (Maximal effect, Emax) of the cells was calculated. The interval and signal-to-noise ratio are shown in Table 2. According to the data in Table 2 and the dose-effect-response curve, select a cell line with a good curve, carry out expanded culture and cryopreserve to establish a cell bank.
[0065] Screening results of table 215 TF-1 monoclonal cell lines
[0066]
[0067] The formula for calculating the signal-to-noise ratio is:
[0068] Signal-to-noise ratio (S / N value) = NGF maximum effect OD value / negative control OD value;
[0069] The formula for calculating the maximum effect interval of a cell is:
[0070] Emax interval = NGF maximum effect OD valu...
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