KC-4601

TMD8-BTK-L528W-KI Cell Line

47767

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Background of TMD8-BTK-L528W-KI Cell Line

Bruton's tyrosine kinase (BTK) is a critical component in B-cell receptor signaling pathways, playing an essential role in B-cell development and function. Mutations in BTK can lead to various immune deficiencies and hematologic malignancies. The L528W mutation in BTK occurs at leucine residue 528, which lies within the kinase domain of the protein. This mutation results in a substitution of leucine with tryptophan, significantly altering the structural integrity and functional activity of BTK.The L528W mutation has been identified in patients with X-linked agammaglobulinemia (XLA), a primary immunodeficiency characterized by a lack of mature B cells and antibodies. Unlike the more commonly studied C481S mutation associated with resistance to BTK inhibitors like ibrutinib, the L528W mutation primarily affects the intrinsic catalytic activity of BTK, leading to reduced phosphorylation of downstream substrates. This impairment in BTK signaling disrupts normal B-cell maturation and function, contributing to severe immune deficiency.Understanding the impact of the L528W mutation on BTK structure and function is crucial for developing targeted therapies for patients with XLA and other related disorders. Further research into this mutation could provide insights into novel therapeutic strategies for managing these conditions.

Specifications

Catalog Number	KC-4601
Cell Line Name	TMD8-BTK-L528W-KI Cell Line
Host Cell Line	TMD8
Description	Stable TMD8 cell line with endogenous BTK-L528W knock-in
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% RPMI-1640+20% FBS+10% DMSO
Propagation Medium	RPMI-1640+10% FBS
Selection Marker	NA
Morphology	lymphoblast
Subculture	Split saturated culture 1:4-1:8 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-L528W-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of TMD8-BTK-L528W-KI Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of TMD8-BTK-L528W-KI Cell Line stable clone using RT PCR sequencing.

Figure 3: Characterization of Dose-response curves and IC50 values for TMD8 and TMD8-BTK-L528W-KI cells treated with Ibrutinib,Pirtobrutinib,Zanubrutinib,Orelabrutinib,BGB-16673 and NX-5948 over 5 days.

Cell Resuscitation

Prewarm culture medium (RPMI-1640 + 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 1:4-1:8 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

Futatani T, Miyawaki T, Tsukada S, Hashimoto S, Kunikata T, Arai S, Kurimoto M, Niida Y, Matsuoka H, Sakiyama Y, Iwata T, Tsuchiya S, Tatsuzawa O, Yoshizaki K, Kishimoto T. Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection. Blood. 1998 Jan 15;91(2):595-602. PMID: 9427714.
2.Lopez-Herrera G, Berron-Ruiz L, Mogica-Martinez D, Espinosa-Rosales F, Santos-Argumedo L. Characterization of Bruton's tyrosine kinase mutations in Mexican patients with X-linked agammaglobulinemia. Mol Immunol. 2008 Feb;45(4):1094-8. doi: 10.1016/j.molimm.2007.07.022. Epub 2007 Aug 31. PMID: 17765309.
3.Nawaratne V, Sondhi AK, Abdel-Wahab O, Taylor J. New Means and Challenges in the Targeting of BTK. Clin Cancer Res. 2024 Jun 3;30(11):2333-2341. doi: 10.1158/1078-0432.CCR-23-0409. PMID: 38578606; PMCID: PMC11147694.