Immortalized Mouse TLR4<sup>-/-</sup> Hepatic Stellate Cells

Mfr.No.	T0689
Description	The Immortalized Mouse Hepatic Stellate Cells were derived from TLR4-/-?mouse via enzymatic digestion and Percoll density gradient centrifugation. The primary cells were immortalized via transfection of the pCMV Simian Virus 40 Large T antigen and hygromycin B selection. The Immortalized Mouse TLR4-/-?Hepatic Stellate Cells are useful in studies focusing on liver fibrosis, especially in the role of the pro-inflammatory TLR4 activation in scar tissue formation. TLR4 lentivirus-mediated knock-in experiments have been performed on Plenti4/TO/V5-DEST GatewayTM vectors (Invitrogen) and lipofectamineTM?2000 (Thermo Fisher Scientific). The cells can be used in studies of cellular survival regulation, as TLR4 activation has been linked to apoptosis-resistance.
Cell Type	Immortalized Cells
Biosafety Level	2
Depositor	ICAHN School of Medicine at Mount Sinai
Disclaimer	1. 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 ?g, Cat.# C207, $450.00) or cell lysate (100 ?g, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final. 2. 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. 3. 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). 4. 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. 5. 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".
Donor History	C57/Bl6 WT mouse
Expression Region	Hygromycin B resistance
Expression Level	Chondrocyte marker COMP, collagen X
Growth Properties	Adherent, polygonal
Growth Conditions	Use of PriCoat?T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. PriGrow III (TM003) + 10% FBS + 1% Penicillin/Streptomycin Solution (G255), 33.0?, 5% CO?
Quantity	1x106 cells / 1.0 ml
Tissue	Liver
Morphology	Multipolar
Notes	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?, 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? or in liquid nitrogen vapor phase. Do not store at -70?, as it will result in loss of viability.
Organism	Mouse (M. musculus)
Population Doubling	Approximately 24 hours
Species	Mouse (M. musculus)
Propagation	Use of PriCoatTM T25 Flasks (G299) or Applied Cell Extracellular Matrix(G422) is required for cell adhesion to theculture vessels. Grow cells in ECM-coated culture vessels unless otherwise specified in the PropagationRequirements below.The base medium for this cell line is Prigrow VIII medium availableat?abm, Cat. No.?TM018. To make the complete growthmedium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentrationof 10% and Penicillin/Streptomycin Solution (G255) to a finalconcentration of 1%.Carbon dioxide (CO2): 5%,Temperature: 37.0°C.* Do not use heat-inactivated FBS for cell culture unless specifiedotherwise.
Cryopreservation	Cryopreservation Medium (TM024), or complete growth media with 10% DMSO.
Quality Control	1) Western blot; 2) qRTPCR; 3) Immunofluorescence

Applications Range	Research Use Only.

Shipping	Ship with dry ice.
Product Format	Frozen
Product 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.
Storage	Vapor phase of liquid nitrogen, or below -130?.

Thawing Protocol	1. Thaw cells quickly in a 37? 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. 1. Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA 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?, for several minutes to facilitate detachment. 3. Neutralize Trypsin-EDTA 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. 6. Incubate the cells at the recommended conditions.

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SKU	Supplier No.	Size	Your price	Quantity
BHC10900846	T0689	1x10^6 cells / 1.0 ml	-		Add to Quote

Immortalized Mouse TLR4 -/- Hepatic Stellate Cells

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