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	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	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.