Description

C57BL/6 Mouse Bone Marrow Macrophages from Cell Biologics are isolated from tibias and femurs of pathogen-free laboratory C57BL/6 mice and grown in tissue culture plates with Cell Biologics’ Cell Culture Medium. Cells at passage 0 are cryo-preserved and each vial contains 3x106 cells per ml. C57BL/6 Mouse Bone Marrow Macrophages are characterized by immunofluorescence staining using antibody of CD11b or F4/80. These cells can be plated on multiwell culture plates for experiments under the cell culture conditions specified by Cell Biologics. Cells can not be cultured indefinitely and can not be passaged.

Suggested Medium

M3368 Complete Macrophage Medium w/ Kit (500 ml)

Primary Macrophage Culture Protocol

All cell culture procedures must be conducted in a bio-safety cabinet.
Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter.
Use aseptic technique to prevent microbial contamination.
Medium Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements).
Use pre-warmed (37°C) cell culture media (30-50 ML, Catalog No. M3368) to seed cells and when changing media.
Coating of Cell Culture Plates or Dishes Coat sterile culture dishes or flasks with Gelatin Coating Solution (Catalog No. 6950, Cell Biologics) for 2 min, then aspirate the excess solution.

Cell Recovery from Cryovial

Thaw the cells quickly (<2 min) in a 37°C water bath until just prior to complete thawing.
Wipe the outside of the vial with 70% ethanol.
Use 1 ml Pipette Tip to resuspend cells and transfer cells to a 15 ml sterile tube.
Add 5 ml of pre-warmed Cell Biologics’ Cell Culture Medium (Catalog number M3368, Cell Biologics) gently to the side of the tube and slowly pipette up and down 2 to 3 times to resuspend the cells.
Centrifuge the cells at 200 x g at 4°C for 5 min.
Carefully remove the supernatant and resuspend in 7 mL pre-warmed Maintenance Medium (Catalog No. M3368, Cell Biologics).
Carefully pour cell suspension into the cell culture plate or dish.

Recommended Cell Seeding: 0.7-0.8 million cells are seeded per well of a 12-well plate or 1-1.5 million macroPlace a plate in a humidified, 5%-CO2 incubator at 37°C until experiments.

Change fresh cell culture medium every 24-48 hours. Cells should be checked daily under a microscope to verify appropriate cell morphology.

Proceed with experiment assays.

Note: Please send us the cell images (>90% confluence) if you have any question or problem with cultured cells.

Storage

Cryopreserved Cells are shipped with dry ice overnight. Upon arrival, transfer frozen cells to liquid nitrogen (-180°C) immediately until ready for use. Live cell shipment is also available on request. Primary cells can never be kept at -20 °C or -80 °C freezer.

Documents

MSDS.CellBiologics

Disclaimer

Investigators should handle the cells with caution and treat all animal cells as potential pathogens, since no test procedure can completely guarantee the absence of infectious agents. Warranty and Liability

Publication

mBio. 2021 May-Jun; 12(3): e01227-21.

mSphere. 2022 Jan-Feb; 7(1): e00934-21.