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Product Name:
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Macrophage Cell Line Derived from TRIF Knockout Mice
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Ownership statement:
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This reagent is the property of the U.S. Government.
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Manufacturer:
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BEI Resources
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Material Provided:
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Each vial contains approximately 1 mL of cell culture suspension frozen in cell growth medium (90%) and DMSO (10%) cryopreservative. Sufficient cells are provided to initiate at least one new culture. The cell count, expressed as cells per vial, is shown on individual certificates of analysis for each product lot.
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Packing/Storage:
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This product was packaged aseptically in cryovials. It should be stored at cryogenic temperature (-100°C or colder), preferably in the vapor phase of a liquid nitrogen freezer. Storage at -70°C will result in loss of viability. To ensure the highest level of viability, the vial should be thawed and the culture initiated as soon as possible upon receipt. Any warming of the product during shipping and transfer must be avoided, as this will adversely affect the viability of the product after thawing. For transfer between freezers and shipping, the cells may be placed on dry ice for brief periods, although use of a portable liquid nitrogen carrier is preferred. Please read the following recommendations prior to reconstituting this material.
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Safety Precautions:
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When handling frozen vials it is highly recommended that protective gloves, lab coat and full face mask be worn. Even brief exposure to the ultra-cold temperature can cause tissue damage from frostbite. Also, some vials may slowly fill with liquid nitrogen if they have been immersed during cryogenic storage. When thawing, the liquid nitrogen may rapidly expand as it changes to gas, breaking the vial or cap with explosive force, sending debris flying with enough velocity to cause injury. Store and use in areas with adequate ventilation.
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Thawing and Growth:
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Prior to thawing the murine macrophage cells, prepare growth medium (GM) for use. Murine macrophage cells are grown in Dulbecco’s Modified Eagle Medium (DMEM; ATCC® 30-2002) modified to contain 10 fetal bovine serum (FBS; ATCC® 30-2020), 4 mM L-glutamine, 1 mM sodium pyruvate and 10 µg/mL ciprofloxacin (optional). This GM is formulated for use with a 5% CO2 in air atmosphere.
Rapidly thaw the vial of murine macrophage cells in a 37°C water bath with gentle agitation. To reduce the risk of contamination, keep the cap and O-ring of the vial out of the water and repeatedly check the cap for tightness during thawing. Remove from the water bath immediately when thawed. Dry the vial with a sterile wiper, decontaminate using a wiper soaked with 70% isopropyl alcohol, and let the vial air dry. Aseptically open the vial, remove the vial contents and add to 4 mL of GM in a centrifuge tube. Centrifuge the cell suspension at 125 to 200 × g for 8 to 10 minutes at 18 to 25°C. Discard the supernatant and resuspend the cell pellet in 10 mL of pre-warmed GM. Transfer the cell suspension into a 75 cm2 tissue culture flask. Incubate the new culture at 37°C and 5% CO2. Replace the GM with fresh GM every 2 to 3 days as needed and incubate until the cell sheet is approximately 80% confluent.
Sub-culture procedure:
Aseptically remove the GM and discard. Briefly rinse the cell layer with 4 to 15 mL of Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline (PBS) to remove all traces of serum. Discard the PBS. Add 2 to 8 mL of 0.05% trypsin-EDTA to the culture flask and incubate the flask at 37°C until cell layer is dispersed (usually within 3 minutes but no longer than 15 minutes).
Note: To avoid clumping, do not agitate the cells by hitting or shaking the flask. Add an equal volume of GM and aspirate cells by gently pipetting. Perform a cell count and add appropriate aliquots of the cell suspension to new culture vessels at a sub-cultivation ratio of 1:3 to 1:4. Adjust the volume of GM to 15 to 20 mL for a 75 cm2 flask. Incubate cultures at 37°C and 5% CO2. Replace the GM with fresh GM every 2 to 3 days and incubate until the cell sheet is approximately 80% confluent.
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Disclaimers:
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You are authorized to use this product for research use only. It is not intended for human use. Use of this product is subject to the terms and conditions of the BEI Resources Material Transfer Agreement (MTA). The MTA is available on our Web site at www.beiresources.org. While BEI Resources uses reasonable efforts to include accurate and up-to-date information on this product sheet, neither ATCC® nor the U.S. Government makes any warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. Neither ATCC® nor the U.S. Government warrants that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, use and disposal. ATCC® and the U.S. Government are not liable for any damages or injuries arising from receipt and/ or use of this product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, the U.S. Government, ATCC®, their suppliers and contributors to BEI Resources are not liable for damages arising from the misidentification or misrepresentation of products. |
References:
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1. Golenbock, D., Personal Communication.
2. Blasi, E., et al. “Selective Immortalization of Murine Macrophages from Fresh Bone Marrow by a raf/myc Recombinant Murine Retrovirus.” Nature 318 (1985): 667-670. PubMed: 4079980.
3. Blasi, E., et al. “A Murine Macrophage Cell Line, Immortalized by v-raf and v-myc Oncogenes, Exhibits Normal Macrophage Functions.” Eur. J. Immunol. 17 (1987): 1491-1498. PubMed: 3119352.
4. Blasi, E., et al. “Generation of Macrophage Cell Line from Fresh Bone Marrow Cells with a myc/raf Recombinant Retrovirus.” Cancer Biochem. Biophys. 10 (1989): 303-317. PubMed: 2695237.
5. Danos, O. and R. C. Mulligan. “Safe and Efficient Generation of Recombinant Retroviruses with Amphotropic and Ecotropic Host Ranges.” Proc. Natl. Acad. Sci. USA 85 (1988): 6460-6464. PubMed: 3413107.
6. Apolloni, E., et al. “Immortalized Myeloid Suppressor Cells Trigger Apoptosis in Antigen-Activated T Lymphocytes.” J. Immunol. 165 (2000): 6723-6730. PubMed: 11120790.
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Citation:
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Acknowledgment for publications should read "The following reagent was obtained through BEI Resources, NIAID, NIH: Macrophage Cell Line Derived from TRIF Knockout Mice, NR-9566."
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Biosafety Level:
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2
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories (BMBL). Current Edition. Washington, DC: U.S. Government Printing Office.
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