I-13.35

The cell line can be used to phenotype and characterize the role of splenic macrophages in the initiation of immune responses.

This is an immortalized cell line derived from the progeny of individual splenic macrophage progenitor cells of an apparently normal adult mouse. Cells were cloned in soft agar in the presence of CSF-1 and expanded on microcarrier beads. Beads were transferred to culture dishes and were subsequently passaged by scraping.

This cell line, unlike I-11.15 (ATCC CRL-2470), has the constitutive ability to present antigen to Th1 helper T cells and it expresses higher levels of MHC Class II antigens than I-11-15. RefMcCormack JM, et al. Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4+ helper T cell subsets and allogeneic CD8+ T cells. Cell. Immunol. 145: 359-371, 1992. PubMed: 1451184

C3H/HeJ strain is defective in TLR4 (toll-like receptor 4)

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes. Discard supernatant.
4. Resuspend the cell pellet with the recommended complete medium and dispense into a 25 cm² culture flask.  It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

1. Remove and discard 75% of culture medium.
2. Scrape cells with cell scraper.
3. Add appropriate aliquots of cell suspension to new culture vessels.
4. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:4 is recommended
Medium Renewal: Every 2 to 3 days

LADMAC Conditioned Medium

LADMAC conditioned medium is made from LADMAC cells (ATCC CRL-2420).  LADMAC cells secrete the growth factor colony stimulating factor 1 (CSF-1). CSF-1 is capable of supporting the in vitro proliferation of mouse bone marrow macrophages. The Pannell-Milstein roller bottle apparatus may be used to produce high concentrations of CSF-1 RefOlivas E, et al. Use of the Pannell-Milstein roller bottle apparatus to produce high concentrations of the CSF-1, the mouse macrophage growth factor. J. Immunol. Methods 182: 73-79, 1995. PubMed: 7769247

Subculturing Procedure for LADMAC cells

Cultures can be established by centrifugation with subsequent resuspension at 2 X 10⁵ viable cells/mL. Attached cells may be dispersed by tapping the sides of the flask until cells are loose.

Complete Growth Medium for LADMAC cells

The base medium for this cell line is ATCC-formulated Eagle's Minimum Essential Medium, Catalog No. 30-2003.  To make the complete growth medium, add the following components to the base medium:  fetal bovine serum to a final concentration of 10%

This medium is formulated for use with a 5% CO₂ in air atmosphere.

Conditioned medium harvesting:

1. Allow LADMAC cells to become confluent.
2. After 5 to 7 days, collect supernate, centrifuge at 125 x g for 5 to 10 minutes
3. Filter (200 nM filter) and tore aliquots at –20oC

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

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Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.