- Medium: We are actively testing different media for their effect on monocyte function. Both serum-free (AIM V™ or X VIVO™ 15) and serum-supplemented media have been evaluated. Human serum at 5–10% in DME/F12 or IMDM have been used at Astarte Biologics with recombinant macrophage colony stimulating factor (M-CSF) added to a final concentration of 10 ng/mL.
- Culture Flasks or Dishes
- Phosphate Buffered Saline without Ca2+ or Mg2+
- Thaw and count cells following standard procedure.
- Adjust cell concentration to 500,000 cells per mL. This concentration can be adjusted based on your protocols.
- Add cells to culture at 1–2.5 x 105 cells per cm2. Incubate at 37°C, 5% CO2.
- Cells can be used for experiments immediately or after 5 days culture.
- If cells are to be maintained for longer periods, the medium should be changed every 3–4 days by removing half the volume of medium and replacing with an equal volume of fresh medium.
Collection of Cells from Culture
- Collect medium from the culture and place in a tube. Immediately add PBS to the adherent cells. Pipet up and down to rinse the culture surface and pool with the cells in medium.
- Add PBS to the culture and place the culture in the incubator at 37°C for 10–15 minutes. During this time, the cells should begin to detach from the culture surface. They become rounder and brighter when observed using a phase contrast microscope.
- If cells are in a flask, the flask can be agitated and rapped against the heel of your hand to dislodge the cells. If using culture dishes or wells, pipet the PBS forcefully against the culture surface to dislodge the cells.
- Collect the cells in PBS and pool with the previously collected cells.
- Monocytes cultured for 5 days in the presence of serum or M-CSF are considered macrophages. Descriptions of monocyte-derived macrophages in scientific literature are usually referring to cells cultured this way.
- Monocytes grow as a mixture of adherent and non-adherent cells with the proportion of adherent cells dependent on the culture medium and activation stimuli (if added). This complicates moving them from one culture vessel to the next or performing experiments that involve collecting intact cells. Recovery of the cells is usually lower than the starting cell number. We recommend setting up the cells in the culture configuration you will use for your experiment. If transfer out of the culture vessel is necessary, use the procedure outlined above.