TLR9 Knockout Immortalized Mouse Dendritic Cell Line

Cat. No.
T3035
Unit
1x10⁶ cells / 1.0 ml
Price
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Cat. No. T3035
Name TLR9 Knockout Immortalized Mouse Dendritic Cell Line
Description


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 MutuDCTlr9-/- cell line is an immortalized splenic CD8α+ subset (CD11chigh,B220-,DEC205+,CD24high,CD11b-) of conventional dendritic cells derived from the TLR9 knockout CD11c:SV40LgT transgenic mice.
The MutuDCTlr9-/- cell line retains response to TLR ligands such as PolyIC (TLR3-L) and to a lesser extent LPS (TLR4-L) but not CpG (TLR9-L). Like the Wild-type MutuDC cells (Cat. No. T0528), these cells are 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) 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.

Organism Mouse (M. musculus)
Tissue Spleen
Donor History TLR3 knockout CD11c:SV40LgT transgenic mice
Growth Properties Adherent, small aggregates
Cell Type Stable Cell Lines
Unit 1x10⁶ cells / 1.0 ml
Storage Condition Vapor phase of liquid nitrogen, or below -130°C.
Shipping Conditions Ship with dry ice.
Product Format Frozen
Intended Use This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
BioSafety II
Certificate of Analysis For batch-specific test results, refer to the applicable certificate of analysis that can be found at www.abmgood.com.
Growth Conditions

Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is recommended for cell adhesion to the culture vessels. Prigrow V (TM015) + 4 mM Glutamax + 10% heat-inactivated FBS + 1% of 7.5% Sodium Bicarbonate Solution + 50 µM beta-mercaptoethanol + 10 mM HEPES (TM058) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 5% CO₂.

Filter the complete media with a 0.22µm filter prior to use. Change the complete media every 2-3 days. Do not let media colour change to yellow.

Unpacking and Storage Instructions

1. Visually examine the packaging containers for signs of leakage or breakage.

2. Immediately transfer frozen cells from dry ice packaging to a temperature below -130°C, preferably in liquid nitrogen vapor phase storage, until ready for use.

To ensure the highest level of viability, thaw the vial and initiate culture as soon as possible upon receipt. If continued storage is desired, the vial should only be stored below -130°C or in liquid nitrogen vapor phase. Do not store at -70°C, as it will result in loss of viability.


Thawing Protocol

1. Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination.

2. Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions.

3. Transfer the cell suspension into a 15ml sterile conical tube containing 5ml of pre-warmed, complete growth media. Centrifuge cells at 125xg for 5-7 minutes.

4. Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media and dispense into a T25 culture flask.

5. Incubate the cells at the recommended conditions.

Subculture Protocol

Volumes given below are for a T75 flask; proportionally increase or decrease the volume as required per culture vessel size. Subculture cells once the culture vessel is 80% confluent. 

Use a non-enzymatic, 5 mM EDTA-based cell dissociation buffer (5 mM EDTA in 20 mM Hepes-PBS) to subculture cells.

1. Aspirate excess media, and add 2-3 ml of the pre-warmed disssociation buffer mixture to the culture vessel,   

2. Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment.

3. Neutralize the solution by adding an equal volume of the complete growth media into the culture vessel.

4. Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type.

5. Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired. Cells must be seeded at high density. 

6. Incubate the cells at the recommended conditions.

Cryopreservation

Cryopreservation Medium (TM024), or complete growth media + 50% FBS + 10% DMSO.

Seeding Density (cells/cm2) 25,000 - 50,000
Split Ratio No greater than 1:3
Population Doubling Time (h) 50 - 60
Expression CD11c, CD24, MHC-II+, DEC205, Clec9A, B220, CD11b, IRF4, IRF8, CD4
Material Citation If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. T3035.
Warranty 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”.
Disclaimer

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

2. All test parameters provided in the CoA are conducted using abm's standardized culture system and 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.

3. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of order placement. 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.

4. 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 or any other non-RUO application(s).

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

6. 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".

Depositor University of Lausanne (PACTT)
Application Research Use Only.
Material Citation If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. T3035
Print & Download Datasheet
  • Fuertes Marraco, S. A., Grosjean, F., Duval, A., Rosa, M., Lavanchy, C., Ashok, D., Haller, S., Otten, L. A., Steiner, Q. G., Descombes, P., Luber, C. A., Meissner, F., Mann, M., Szeles, L., Reith, W., & Acha-Orbea, H. (2012). Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research. Frontiers in immunology, 3, 331. https://doi.org/10.3389/fimmu.2012.00331

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