KC-4701

TMD8-BTK-V416L-KI-1B1 Cell Line

51877

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Background of TMD8-BTK-V416L-KI-1B1 Cell Line

BTK-V416L is a specific mutation found in the Bruton's tyrosine kinase (BTK) gene, which encodes a member of the Tec family of non-receptor tyrosine kinases. BTK plays a critical role in B-cell development, activation, and survival by transducing signals from the B-cell receptor (BCR) to downstream effectors. The V416L mutation involves the substitution of valine for leucine at position 416 within the kinase domain of BTK, potentially altering its senzymatic activity and substrate specificity.Studies have shown that the BTK-V416L mutation can result in a conformational change in the kinase domain, affecting both the binding affinity of ibrutinib and the catalytic efficiency of BTK. This alteration can lead to sustained BCR signaling despite the presence of the inhibitor, contributing to disease progression. Understanding the molecular mechanisms underlying this resistance is crucial for developing alternative therapeutic strategies for patients who develop resistant mutations.

Specifications

Catalog Number	KC-4701
Cell Line Name	TMD8-BTK-V416L-KI-1B1 Cell Line
Host Cell Line	TMD8
Description	Stable TMD8 clone expressing endogenous BTK gene bearing V416L mutations, No.1B1
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	N/A
Morphology	lymphoblast
Subculture	Split saturated culture 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

TMD8-BTK-V416L-KI-1B1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of TMD8-BTK-V416L-KI-1B1 cell line stable clone using PCR sequencing.

Figure 2: Characterization of TMD8-BTK-V416L-KI-1B1 cell line stable clone using RT-PCR sequencing.

Figure 3. Characterization of dose-response curves for BTK inhibitors on TMD8 and TMD8-BTK-V416L-KI-1B1 cells.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Woyach, J. A., et al. (2014). Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib.New England Journal of Medicine, 370(25), 2286-2294. https://doi.org/10.1056/NEJMoa1400077
2.Brown, J. R., et al. (2016). Preclinical characterization of acalabrutinib (ACP-196): a next-generation selective Bruton tyrosine kinase inhibitor.Cancer Discovery, 6(3), 325-334. https://doi.org/10.1158/2159-8290.CD-15-0828
3.Kahl, B. S., et al. (2017). Ibrutinib versus temsirolimus in patients with relapsed or refractory mantle-cell lymphoma: interim analysis results of an open-label, phase 3 trial.The Lancet Oncology, 18(8), 1017-1028. https://doi.org/10.1016/S1470-2045(17)30429-5