KC-4533

TMD8-BTK-A428D-KI-1B4 Cell Line

52203

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Background of TMD8-BTK-A428D-KI-1B4 Cell Line

Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor (BCR) signaling pathway, playing a pivotal role in B-cell development, differentiation, and survival. BTK mutations have been implicated in various B-cell malignancies, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström's macroglobulinemia (WM). Among these mutations, BTK-A428D has emerged as a significant point mutation associated with resistance to covalent BTK inhibitors (BTKis), such as ibrutinib and acalabrutinib. This mutation occurs at the adenosine triphosphate (ATP)-binding site of BTK, leading to reduced binding affinity of these inhibitors and subsequent therapeutic resistance.The BTK-A428D mutation is characterized by the substitution of alanine with aspartic acid at position 428, a residue located in the kinase domain of BTK. This alteration disrupts the covalent binding of irreversible BTK inhibitors, which typically target the cysteine residue at position 481 (C481). As a result, the mutated BTK retains its kinase activity, allowing malignant B-cells to proliferate despite treatment. The emergence of BTK-A428D and other resistance mutations underscores the need for next-generation BTK inhibitors that can overcome these mechanisms of resistance.

Specifications

Catalog Number	KC-4533
Cell Line Name	TMD8-BTK-A428D-KI-1B4 Cell Line
Host Cell Line	TMD8
Description	Stable TMD8 clone expressing endogenous BTK gene bearing A428D mutations, No.1B4
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	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-A428D-KI-1B4 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of TMD8-BTK-A428D-KI-1B4 cell line stable clone using PCR sequencing..

Figure 2: Characterization of TMD8-BTK-A428D-KI-1B4 cell line stable clone using RT-PCR sequencing..

Figure 3: Characterization of dose-response curves for BTK inhibitors on TMD8 and TMD8-BTK-A428D-KI-1B4 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% RPMI-1640 + 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

Estupiñán HY, Berglöf A, Zain R, Smith CIE. Comparative analysis of BTK inhibitors and mechanisms underlying adverse effects. Front Cell Dev Biol. 2021;9:630942. doi:10.3389/fcell.2021.630942.
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.
Woyach JA, Furman RR, Liu TM, et al. Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370(24):2286-2294. doi:10.1056/NEJMoa1400029.