J774A.1 Cell Culture

ATCC: TIB-67

Link: https://www.atcc.org/Products/All/TIB-67.aspx

Organism: Mouse

Morphology: Monocyte/Macrophage

Culture: Adherent

BSL: 1

Culture Medium: DMEM + 10% Fetal Bovine Serum and 1% Pen/Strep

Passaging conditions: Change medium for fresh, pre-warmed medium and scrape with sterile cell scraper. Resuspend in fresh medium.

Cryopreservation medium: pH-equilibrated culture medium + 10% ultra-pure DMSO

The cells should exist as an almost 50/50% population of small, round, monocyte-like cells as well as stretched bi-directional, fibroblast-like cells. If the cells become stellate (star-like) and highly vacuolar then they are stressed. Return the flask to the incubator and monitor daily for phenotype improvement, changing the medium as necessary. Do not passage the cells if they are stressed, as they will die (<60% viable). Do not use medium that appears highly alkaline (deep pink) when removed from the culture fridge – this usually happens when a bottle of medium is close to 2 months old.

Do not trypsinize these cells.

Highly sensitive and virtually untransfectable with expression plasmids without virus or nucleofection.

Strongly adherent, no ECM coating necessary. These cells are extremely sensitive to neighboring cell damage. While scraping for passaging, ensure the cells are thoroughly covered in medium by angling the flask.

Pro-inflammatory (M1) priming can be performed by stimulating the cells with LPS or other TLR stimulants. E.g., 1 ug/mL E. coli O111:B4 LPS for 4 hours or overnight. The cells will produce IL-1beta only when primed, but produce IL-18 constitutively.