KC-5482

TMD8-BTK-M437R-KI Cell Line

58019

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Background of TMD8-BTK-M437R-KI Cell Line

Bruton’s tyrosine kinase (BTK) is a key regulator of B-cell receptor signaling and is critically involved in B-cell survival and proliferation. The M437R mutation in BTK, resulting from a methionine-to-arginine substitution at position 437 within the kinase domain, has been identified as a mechanism of resistance to certain non-covalent BTK inhibitors. Unlike the canonical M437R mutation that abrogates covalent binding, M437R induces steric constraints and alters the physicochemical properties of the ATP-binding pocket, thereby reducing drug affinity while often preserving kinase activity. This mutation highlights the evolving landscape of inhibitor resistance and underscores the need for structural-guided drug design to develop agents effective against such resistant variants.

Specifications

Catalog Number	KC-5482
Cell Line Name	TMD8-BTK-M437R-KI Cell Line
Clone Number	1C1
Host Cell Line	TMD8
Description	Stable TMD8 clone expressing exdogenous BTK gene bearing M437R mutations, No.1C1
Quantity	One vial 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	NA
Morphology	Lymphoblast
Subculture	Split saturated culture 1:3-1:4 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-M437R-KI-1C1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of TMD8-BTK-M437R-KI cell line stable clone using PCR sequencing..

Figure 2: Characterization of TMD8-BTK-M437R-KI cell line stable clone using RT-PCR sequencing..

Figure 3: Characterization of dose-response curves for BTK inhibitors on TMD8-BTK-M437R-KI cells.

Cell Resuscitation

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

Cell Freezing

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

References

Bond DA, Woyach JA. Targeting BTK in CLL: beyond ibrutinib. Curr Hematol Malig Rep. 2019;14(3):197-205. doi:10.1007/s11899-019-00512-0.
Wang E, Mi X, Thompson MC, et al. Mechanisms of resistance to noncovalent Bruton's tyrosine kinase inhibitors. N Engl J Med. 2022;386(8):735-743. doi:10.1056/NEJMoa2114110.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.