Immortalized Mouse Dendritic Cells (MutuDC1940)

As messengers bridging the innate and adaptive immune system, the conventional tissue-resident DC (cDC) acts as sentinels in secondary lymphoid organs and other tissues for antigen capture and presentation. The Wild-type MutuDC1940 cell line is an immortalized splenic CD8α⁺ subset (CD11c^high,B220^-,DEC205⁺,CD24^high,CD11b^-) of conventional dendritic cells derived from the CD11c:SV40LgT transgenic mice.
The MutuDC1940 cell line retains response to TLR ligands such as CpG (TLR9-L) and PolyIC (TLR3-L) and to a lesser extent LPS (TLR4-L) stimulation by up-regulation of co-stimulatory molecules CD40, CD80 and CD86. In addition to responding to PAMP stimulation and producing Th1 cytokines such as IL-12, these cells are also capable of presenting antigen in the context of both MHC-I and MHC-II, including direct antigen presentation and cross-presentation of cell-associated antigens. Together with TLR3 knockout (Cat. No. T3034), TLR9 knockout (Cat. No. T3035) and Ifnar1 knockout (Cat. No. T3036) MutuDC, these dendritic cell lines provide a powerful tool in vaccine science and immunotherapy, particularly in strategizing target antigens to CD8α⁺ subset.

Immortalization Method: Isolated from C57BL/6 transgenic mice carrying SV40 Large T oncogene

BioSafety Level: II

Species: Mouse

Source Organ: Spleen

Growth Properties: Adherent

Morphology: Small aggregates

Population Doubling: 45 - 55 hours

Seeding Density: 20,000 – 60,000 cells/cm². Recommended split ratio:no greater than 1:3.

Markers: CD11c, CD24, MHC-II+, DEC205, Clec9A, B220, CD11b, IRF4, IRF8, CD4

Donor Age: N/A

Donor Gender: N/A

Donor Ethnicity: N/A

Propagation: The base medium for this cell line is IMDM (1x) + Glutamax^TM (Gibco Ref: 31980-030). To make the complete growth medium, add the following components to the base medium: heat-inactivated fetal bovine serum (TM999) to a final concentration of 10%, 1% of 7.5% Sodium Bicarbonate Solution, 50 µM β-mercaptoethanol, HEPES to a final concentration of 10 mM, and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%. Filter the complete media at 0.22µm before use.
Carbon dioxide (CO₂): 5%, Temperature: +37.0°C.



To decomplement FBS:

1. Let the FBS bottle completely thaw overnight at 4°C.

2. Leave the FBS bottle in water bath for 30 minutes at 56°C.

3. Decomplemented FBS can be stored at -20°C for long term. Avoid frequent freeze-thaw cycling.



Change the complete media every 2-3 days. Do not let media colour change to yellow.



To thaw T0528:

1. Thaw the vial in 37^oC water bath until there is no more than a small cube of ice.

2. Transfer the cells to a 15ml tube containing 5ml of pre-warmed complete media.

3. Centrifuge the cells at 290xg for 5 minutes.

4. Carefully discard supernatant without disturbing the cell pellet and gently resuspend the cells in 1ml of complete media by lightly pipetting up and down.

5. Seed the cells at 20,000 - 60,000 cells/cm².


To subculture T0528:

1. It is recommended to subculture when cells are at 70-90% confluency.

2. Aspirate old media. Some cells in suspension are still viable cells. Alternatively,
the supernatant containing the suspended cells can also be collected and centrifuged (Skip to Step 6 in protocol).


3. Add 1:1 ratio of 1X sterile PBS and 0.25% Trypsin-EDTA (TM051).
4. Incubate cells at room temperature for 3-5 minutes and agitate the culture vessel until 90% of
the cells have detached.

5. Immediately neutralize the trypsin by adding complete media equal to the volume of trypsin +
PBS added.

6. Collect the cells and centrifuge at 290xg for 5 minutes.

7. Discard the supernatant and gently resuspend the cells in complete media by lightly pipetting up
and down.

8. Recommended split ratio is no more than 1:3.


Stimulation can be performed using PolyIC (5 µg/ml), CpG (2 mM) or LPS (5 µg/ml). These cells are especially sensitive to FBS requirement, thus, it is advised to use the same batch for culturing cells that show best result in supporting their culture. We also recommend the addition of extra 1% Hepes to the complete media to encourage propagation of the cells.


To freeze T0528:

Recommended freezing medium:

Complete growth medium with decomplemented FBS to a final concentration of 50% and 5% DMSO.

Storage temperature: Liquid nitrogen vapour phase.

Quality Control: 1) Direct antigen presentation and cross-antigen presentation were evaluated by the MHC-I (SIINFEKL/OT-I ) and MHC-II (OVA323-339/OT-II) restricted systems; 3) proteome profile and surface markers assessed by RT-PCR; RT-PCR; 3) IL-12 cytokine secretion analyzed using ELISA ; 4) Response to PAMP stimulation evaluated by functional assays.

Shipping Condition: Dry Ice

Storage Condition: liquid nitrogen or -180°C