Primary Human Macrophages

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.

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Protocol:

Phlebotomy & Mononuclear cell Isolation:

1. 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).
2. 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.
3. Centrifuge at 500 x g for 25 minutes, with the centrifuge acceleration set to 5 and the deceleration set to 0.
4. 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.
5. 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:

1. 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.
2. 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).
3. 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.
4. Carefully transfer to the incubator, and incubate at 37C/5% CO₂ for 1 hour.  During this time monocytes will adhere to the coverglasses.
5. Remove non-adherent cells (T, B & NK cells) by washing each well twice with 37C PBS.
6. Add 1ml of culture media to each well, with the appropriate cytokines added for the desired differentiation state.

Non-differentiated (M0) Macrophages:

1. Immediately after step 7 in the 'plating cells' section add 10ng/ml of recombinant human M-CSF (rhM-CSF)
2. Culture for 5-7 days, exchanging the media & cytokines if required.

Classically Activated (M1) Macrophages:

1. Immediately after step 7 in the 'plating cells' section add 20ng/ml of recombinant human GM-CSF (rhGM-CSF)
2. Culture for 5 days, exchanging the media & cytokines if required.
3. On day 5 replace media and add 20ng/ml rhGM-CSF + 250ng/ml LPS + 10ng/ml INFγ (*stock is 10,000x).
4. Culture an additional 2 days.

Alternatively Activated (M2) Macrophages:

1. Immediately after step 7 in the 'plating cells' section add 10ng/ml of recombinant human M-CSF (rhM-CSF)
2. Culture for 5 days, exchanging the media & cytokines if required.
3. On day 5 replace the media and add 10ng/ml rhM-CSF + 10ng/ml of rhIL-4.
4. Culture an additional 2 days.