KC-2104
MC38-MR1-Cell-Line
Background of MC38-MR1-Cell-Line
MR1 (Major Histocompatibility Complex, Class I-Related) also known as HLALS, it is a Protein Coding gene. The protein encoded by this gene is an antigen-presenting molecule that presents metabolites of microbial vitamin B to MAITs. Mucosa-associated invariant T (MAIT) cells are MR1-restricted, innate-like T lymphocytes with tremendous antibacterial and immunomodulatory functions. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. Diseases associated with MR1 include Immune Deficiency Disease and Hemochromatosis, Type 1.
Specifications
| Catalog Number | KC-2104 |
|---|---|
| Cell Line Name | MC38-MR1-Cell-Line |
| Host Cell Line | Mouse MC38 cell line |
| Description | Stable MC38 clone expressing exogenous MR1 gene. |
| 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% DMEM + 20% FBS + 10% DMSO |
| Propagation Medium | DMEM + 10% FBS + 5μg/mL Puromycin |
| Selection Marker | Puromycin |
| Morphology | Epithelial |
| Subculture | Split saturated culture 1:2-1:5 every 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 48 hours |
| Mycoplasma Status | Negative |
| In Vivo Validation | NA |
Cell Line Generation
MC38-MR1 cell line was generated using a lentiviral vector expressing the MR1 sequence.
Characterization
Figure 1: Characterization of MR1 overexpression in the MC38-MR1 stable clone using QPCR.
Cell Resuscitation
- Prewarm culture medium (DMEM supplemented with 10% FBS and 5μg/mL puromycin)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 1:2-1:5 every 3 days; seed out at about 1-3 × 105 cells/mL.
Cell Freezing
- Prepare the freezing medium (70% DMEM + 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
- Lim HJ, Wubben JM, Garcia CP, Cruz-Gomez S, Deng J, Mak JYW, Hachani A, Anderson RJ, Painter GF, Goyette J, Amarasinghe SL, Ritchie ME, Roquilly A, Fairlie DP, Gaus K, Rossjohn J, Villadangos JA, McWilliam HEG. A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells. J Cell Biol. 2022 Dec 5;221(12):e202110125. doi: 10.1083/jcb.202110125. Epub 2022 Sep 21. PMID: 36129434; PMCID: PMC9499830.
- Lepore M, Lewinsohn DA, Lewinsohn DM. T cell receptor diversity, specificity and promiscuity of functionally heterogeneous human MR1-restricted T cells. Mol Immunol. 2021 Feb;130:64-68. doi: 10.1016/j.molimm.2020.12.009. Epub 2020 Dec 23. PMID: 33360378; PMCID: PMC7855563.
- McSharry BP, Samer C, McWilliam HEG, Ashley CL, Yee MB, Steain M, Liu L, Fairlie DP, Kinchington PR, McCluskey J, Abendroth A, Villadangos JA, Rossjohn J, Slobedman B. Virus-Mediated Suppression of the Antigen Presentation Molecule MR1. Cell Rep. 2020 Mar 3;30(9):2948-2962.e4. doi: 10.1016/j.celrep.2020.02.017. PMID: 32130899; PMCID: PMC7798347.