J774 Macrophage Cell Culturing

  1. J774 cell line, a murine macrophages cell line established from a tumor that arose in a female BALB/c mouse, were provided by Prof. Yi-Hsuan Lee from National Yang Ming University.
  2. View cultures using a microscopy and after the cultures was full of the dish, remove the spent medium.
  3. Wash the cell monolayer with PBS and remove it.
  4. Add medium(1ml per 25 cm2 of surface area) and scrape cells down.
  5. Ensure cells were detached by a microscopy.
  6. Centrifuge under 2000 rpm, 5minutes, 25°C.
  7. Remove the supernatant and add 5 ml medium to suspend cells.
  8. Count the cells by Bright Line Counting Chamber. Subject the cell diversity to 104/ml.
  9. Seed the cells for 104/well, 1ml/well in the 24-well plate.
  10. Until cells grow full of each well, do the coculturing experiment.