Immortalized Mouse Dendritic Cells (MutuDC1940)

CAT.NOUNITPRICE
T05281x106 cells / 1.0 ml
$850.00

Specifications


Description

As messengers bridging the innate and adaptive immune systems, conventional/classical lymphoid tissue-resident dendritic cells (cDCs) capture and present antigens, acting as sentinels in secondary lymphoid organs and other tissues. The wild-type MutuDC1940 dendritic cell line is derived from mouse spleen tissues (CD11c:SV40LgT transgenic mice) and belongs to the CD8α+ subset (which has the CD11chigh, B220, DEC205+, CD24high, CD11b expression profile). MutuDC lines are GFP positive due to the GFP reporter in the CD11c:SV40LgT transgene. This cell line has the following characteristics:

  • responds to TLR ligands such as CpG (TLR9-L), PolyIC (TLR3-L), and LPS (TLR4-L) by up-regulation of CD40, CD80 and CD86
  • responds to PAMP stimulation by production of Th1 cytokines such as IL-12
  • capable of MHC-I and MHC-II antigen-presentation (both direct and cross-presentation of cell-associated antigens)

The MutuDC1940 dendritic cell line is a powerful tool for vaccine science and immunotherapy, particularly for strategizing target antigens to the CD8α+ subset. In addition, our catalog contains the following MutuDC cell lines:

SKUT0528
SpeciesMouse (M. musculus)
Species descriptionCD11c:SV40LgT-transgenic C57BL/6 mice
Tissue/Organ/Organ SystemSpleen
Growth PropertiesAdherent
Cell MorphologySmall aggregates
Seeding Density40,000 – 60,000 cells/cm2.

Recommended split ratio: no greater than 1:3.
Population Doubling Time45 - 55 hours
Immortalization MethodIsolated from C57BL/6 transgenic mice carrying SV40 Large T oncogene
Applications

For Research Use Only

Unit quantity1x106 cells / 1.0 ml
Cell TypeImmortalized Cells
Expression Profile

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

Propagation Requirements

The base medium for this cell line is IMDM (1x) + GlutamaxTM (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.
Change the complete media every 2-3 days. Do not let media colour change to yellow.
Carbon dioxide (CO2): 5%, Temperature: 37.0°C.

To heat-inactivate 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. Heat-inactivated FBS can be stored at -20°C for long term. Avoid frequent freeze-thaw cycling.

To thaw T0528:
1. Thaw the vial in 37°
C 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/cm
2.

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 heat-inactivated FBS to a final concentration of 50% and 5% DMSO.
Storage temperature: Liquid nitrogen vapour phase.

QC

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

Disclaimer

1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item.

2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected].

3. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.

4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).

5. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.

6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.

7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period."

DepositorUniversity of Lausanne (PACTT)
FAQs


I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock).
How often do I need to change the media?
The media should be changed every 2-3 days.
Why do these cells need bio safety level II?
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required.
Do you sell ECM coated T75 flasks?
Yes we can provide a coating service. Please inquire with [email protected]
What can I coat a larger dish to subculture?
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html
How long can I store frozen vials for?
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery.
Should the cap of the flask be changed before starting the cell culturing step?
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.).
What is the recommended storage temperature?
In general, if you received: Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen; -180C.
What is the growth rate?
The cells grow to 80-90% confluence in 3-4 days. Recommended Seeding Density: 2.5 x 105 cells/cm2, should not be split lower than 5 x 104 cells per cm2.
How is cell density crucial for drug selection?
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don't need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic.
My cells are not detaching, what method do you recommend to trypsinize the cells?
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope. 2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells. 3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
Why is it important to determine the optimal seeding density?
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
References


14
  • Fuertes Marraco, SA et al. "Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research" Front Immunol 3:331 (2012). DOI: 10.3389/fimmu.2012.00331.
  • Duval, A et al. "Large T Antigen-Specific Cytotoxic T Cells Protect Against Dendritic Cell Tumors through Perforin-Mediated Mechanisms Independent of CD4 T Cell Help" Front Immunol 5:338 (2014). DOI: 10.3389/fimmu.2014.00338.
  • Marraco, SA et al. "Novel Murine Dendritic Cell Lines: A Powerful Auxiliary Tool for Dendritic Cell Research" Front Immunol 3:331 (2012). DOI: 10.3389/fimmu.2012.00331.
  • Fuertes Marraco , SA et al. "Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research" Front Immunol. 3:331 (2012). PubMed: 23162549.
  • Joshi, PS et al. "Characterization of immortalized human mammary epithelial cell line HMEC 2.6" Tumour Biol. 39(10):1010428317724283 (2017). DOI: 10.1177/1010428317724283. PubMed: 29022488.