TLR9 Knockout Immortalized Mouse Dendritic Cell Line

Cat. No.

T3035

Unit

1x10⁶ cells / 1.0 ml

Price

Inquiry

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Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing institution.

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Specifications

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 (CD11c^high,B220^-,DEC205⁺,CD24^high,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.
Cat. No.	T3035
Name	TLR9 Knockout Immortalized Mouse Dendritic Cell Line
Unit	1x10⁶ cells / 1.0 ml
Price	Inquiry
Category	Stable Cell Lines
Cell Type	Stable Cell Lines
Organism	Mouse (M. musculus)
Tissue	Spleen
Donor History	TLR3 knockout CD11c:SV40LgT transgenic mice
Cell Morphology	Small Aggregates
Growth Properties	Adherent, small aggregates
Seeding Density (cells/cm²)	25,000 - 50,000
Split Ratio	No greater than 1:3
Population Doubling Time	50 - 60
Expression Profile	CD11c, CD24, MHC-II+, DEC205, Clec9A, B220, CD11b, IRF4, IRF8, CD4
Expression	CD11c, CD24, MHC-II+, DEC205, Clec9A, B220, CD11b, IRF4, IRF8, CD4
Propagation Method	The base medium for this cell line is PriGrow V available at abm (TM015). To make the completed growth medium, add the following components to the base medium: 10% decomplemented fetal calf serum (PAN biotech; P40-37500), 50 µM ?-mercaptoethanol, 1% HEPES, and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%. Stimulation can be performed using CpG (2 mM) or LPS (5 µg/ml). Carbon dioxide (CO₂): 5%, Temperature: 37.0°C. IMPORTANT: This cell line is very sensitive to FBS batches. We strongly recommend end users to use FBS obtained from PAN biotech.
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.
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.
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.
Application	Research Use Only.
Storage Condition	Vapor phase of liquid nitrogen, or below -130°C.
BioSafety	II
Caution	For Research Use Only
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 licensing@abmgood.com. 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)

Documents

Supporting Protocol

Other

Cell Line Catalog (Volume 2)

STR

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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 perform extensive downstream characterization, or gene expression profiling of our cell lines. However, you can check the ‘References’ tab to see if a publication is available, or you can contact us to see if RNA or cell lysates are available for your cell line of interest. Please directly contact us for further information at quotes@abmgood.com.

	Does abm's PriGrow series medium contain antibiotics?
	No, the medium does not contain antibiotics and needs to be supplemented by the end-user as desired.

	Do I need to use abm's media and Applied Cell Extracellular Matrix (G422) to culture my cells?
	We strongly encourage using the recommended media and culture conditions described in the "Growth Conditions" section as they have been optimized for cell growth. Other supplier's media and coating conditions have not been tested.

	What is the difference between Applied Cell Extracellular Matrix (G422) and Collagen Coating?
	The main component of our Applied Cell Extracellular Matrix (G422) is type I collagen specifically. For more information on abm's Applied Cell Extracellular Matrix please visit: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html.

	Do I have to use T25 ECM-coated flasks for growing the cells?
	We strongly recommend thawing cells in the T25 flasks specified on the cell-specific product page to ensure optimal post-thaw recovery of the frozen cells before testing other culture vessels.

	If I want to plate these cells to multi-well plates (e.g. 96 well plates) or dishes, how should I prepare the plates?
	For a protocol on how to coat plates and dishes with Applied Cell Extracellular Matrix (Cat. No. G422), please download the “Applied Cell Extracellular Matrix Data Sheet” from here.

	What percentage of Trypsin-EDTA should be used to subculture cells? Can I use Trypsin-EDTA containing phenol red?
	The standard concentration used for subculturing is 0.25% Trypsin-EDTA, available at abm (Cat. No. TM050). Yes, Trypsin-EDTA containing phenol red can be used as it does not interfere with trypsinization.

	How often do I need to change the media?
	Media should be changed every 2-3 days or as specified within the recommended growth conditions.

Why are these cells classified as biosafety level II?

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 desired.

	How long can I store frozen vials?
	Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen vapor phase (or below -130°C) indefinitely without affecting viability.

What is the recommended storage temperature for cells?

In general, if you received:

Live cells: acclimatize for 3-5 hours at the recommended temperature and CO2 conditions stated for the cell line under the Growth Conditions section, and then change media afterwards. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).
Frozen cells: Upon receipt, immediately place cells in liquid nitrogen; or below -130°C.

	Can I substitute heat-inactivated FBS with FBS or vice versa?
	FBS and heat-inactivated FBS are different in their composition; they cannot be substituted for one another.

My cells are not detaching, what method do you recommend to trypsinize the cells?

Gently wash adherent cells with PBS (1X, without calcium or magnesium) to remove trypsin inhibitors before adding the trypsinization agent.
Ensure the correct trypsin concentration is being used (i.e. 0.25%).
Do not use cold trypsin to detach cells as the proteolytic activity will be low. Pre-warm trypsin to 37°C for optimal enzymatic activity.
Incubate the cells with trypsin-edta agent at 37°C to facilitate cell detachment.
Gently tap the side of the culture vessel to facilitate mechanical detachment.

How are live cells shipped?

Live cells are shipped as a live culture in a T12.5 flask. Cells are seeded at an optimal confluency to avoid metabolic waste build up during transport. In addition, we thoroughly test our cell lines for functional freeze-thaw recovery. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).

	Why are cells not attaching and forming clumps after subculturing with trypsin?
	Cells form clumps and display attachment issues when the trypsinization agent is not effectively neutralized. To completely neutralize the trypsin agent, add an equal amount of complete growth media prior to centrifugation. Subtle changes in culture conditions, particularly in pH and the quality of serum used in the growth medium, may also affect the amount of clumping exhibited by the cells.

	Why are my cells forming clumps after plating?
	It is important to ensure cell clumps are broken up through gentle re-suspension by pipetting up and down several times after pelleting cells by centrifugation. This will ensure a homogenous single cell suspension for even plating.

	Do I need to add the selection drug to the complete growth medium?
	We recommend maintaining selection pressure using the drug concentration specified in the Growth Conditions section.

	Where can I find the CoA for my product?
	Certificates of analysis can be found here (https://www.abmgood.com/CoA-Library.html).

References

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.

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