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	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	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.

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.