Primary Human Macrophages
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Isolation & Culturing Primary Human Macrophages
Human macrophages can be prepared from anticoagulant-treated peripheral blood mononuclear cells. By culturing monocytes with different cytokine cocktails, undifferentiated monocyte-derived macrophages (M0/MDM), M1 polarized or M2 polarized cells can be produced.
Recipes:
ACD (optional):
To 250ml of ddH2O add:
- 7.36 g Citric Acid
- 14.71 g Sodium Citrate
- 9.91 g Dextrose
Culture Media:
- Culture media is RPMI 1640 media supplemented with 10% FBS and antibiotics/antimycotics at the manufacturers recommended concentration.
Protocol:
Phlebotomy & Mononuclear cell Isolation:
- Draw human blood and immediately transfer to a tube containing an appropriate anticoagulant (10ul/ml of 1000U/ml heparin, 1ml ACD/5ml blood, etc). Mix well. Draw ~7.5ml of blood for every 12-well plate of macrophages you wish to prepare (e.g. 30ml of blood is sufficient for 4 x 12-well plates).
- Layer blood in 15ml tubes over an equal volume of Lympholyte-poly (Cedarlane) as per manufacturers instructions, being careful to not disturb the interface between the blood and lympholyte-poly.
- Centrifuge at 500 x g for 25 minutes, with the centrifuge acceleration set to 5 and the deceleration set to 0.
- Carefully remove the tubes from the centrifuge and use a pipette to carefully remove the top band of cells (mononuclear fraction). If needed, the second band (neutrophil fraction) can also be removed.
- Wash isolated mononuclear fraction in room-temperature PBS, 1500 RPM (300G), 6 minutes. Full acceleration and break can be used for this step.
Plating Cells:
- During the mononuclear cell isolation, add sterile 18mm circular coverglasses to each well of the 12-well plates. Warm cover-glass loaded plates in the incubator while completing mononuclear cell isolation.
- Suspend the washed mononuclear isolate from step 5, above, in 200ul of culture media per well of cells being plated (e.g. 2.4ml would be used for a single 12-well plate).
- Mix gently, then pipette 200ul of the mononuclear suspension into the centre of each coverglass. The cell suspension should form a 'bubble' on the coverglass and not be allowed to spread across the whole well. Adding cells in this fashion will maximize the number of usable cells on each coverglass.
- Carefully transfer to the incubator, and incubate at 37C/5% CO2 for 1 hour. During this time monocytes will adhere to the coverglasses.
- Remove non-adherent cells (T, B & NK cells) by washing each well twice with 37C PBS.
- Add 1ml of culture media to each well, with the appropriate cytokines added for the desired differentiation state.
Non-differentiated (M0) Macrophages:
- Immediately after step 7 in the 'plating cells' section add 10ng/ml of recombinant human M-CSF (rhM-CSF)
- Culture for 5-7 days, exchanging the media & cytokines if required.
Classically Activated (M1) Macrophages:
- Immediately after step 7 in the 'plating cells' section add 20ng/ml of recombinant human GM-CSF (rhGM-CSF)
- Culture for 5 days, exchanging the media & cytokines if required.
- On day 5 replace media and add 20ng/ml rhGM-CSF + 250ng/ml LPS + 10ng/ml INFγ (*stock is 10,000x).
- Culture an additional 2 days.
Alternatively Activated (M2) Macrophages:
- Immediately after step 7 in the 'plating cells' section add 10ng/ml of recombinant human M-CSF (rhM-CSF)
- Culture for 5 days, exchanging the media & cytokines if required.
- On day 5 replace the media and add 10ng/ml rhM-CSF + 10ng/ml of rhIL-4.
- Culture an additional 2 days.