General protocol for the culture of Primary Macrophages

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.

Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival. 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) to recover cryo-preserved cells or when changing media or splitting cells.

Cell recovery from cryovial:

• Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.
• Promptly remove the vial and wipe it down with 70% ethanol.
• Transfer cells from the vial to a sterile centrifuge tube. Add 10 ml of pre-warmed Cell Biologics Cell Culture Medium. Flush the vial with an additional 0.5-1.0 ml of medium to ensure complete transfer of cells to the centrifuge tube.
• Centrifuge cells at 100 g for 5 minutes.
• Aspirate the supernatant and resuspend the cell pellet in 5-7 ml of Cell Biologics’  Cell Culture Growth Medium.
• Add resuspended cells into a plate (tissue culture treated).

Recommended Cell Seeding:

• 0.7-0.8 million cells are seeded per well of a 12-well plate or 1-1.5 million macrophages are seeded per well of a 6-well plate.
• Place 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 microscopy to verify appropriate cell morphology.

Thank you!

Cell Biologics