How to: Isolation of primary Monocytes

While immortalized cell lines offer convenience in many experiments, there are instances where they fall short. Take, for example, monocytes — cell lines like THP-1, while commonly used, are limited as they exclusively express CD14+ and lack CD16 expression. This posed a challenge when validating our hypothesis on CD16-mediated, antibody-dependent infection of SARS-CoV-2. To address this, we turned to primary monocytes sourced from healthy donor blood. Additionally, when delving into the immune cells of patients, the isolation of primary monocytes becomes crucial for accurate insights.

Reagents

• Apheresis leukoreduction collar or whole blood
• PBS
• Fetal calf serum (FCS/ FBS)
• Ficoll Paque Plus (GE Healthcare, #GE17–1440–02)
• CD14 MicroBeads human (Miltenyi Biotec, #130–050–201)
• MACS LS columns & separator
• RPMI 1640
• Human serum albumin
• TC-supplements (10 U/ml Penicillin G, 100 ug/ml Streptomycin, 6 mM Acid-free HEPES, 1.6 mM L-glutamine, 50 uM beta-mercaptoethanol)
• LPS

All vendors are suggestions and are provided to look up manufacturer protocols.

Protocol

1. Drain the collar (approx. 10 ml) into a 50 ml tube when using a leukoreduction collar. Dilute it 1:3 with PBS to reinstate the volume of whole blood (approx. 30 ml). This step can be skipped if using whole blood.
2. Layer the blood on top of 15 ml Ficoll. Therefore fill 15 ml Ficoll into a 50 ml tube, tilt the falcon until the liquid surface is almost horizontal, and then slowly let the blood flow along the wall of the falcon. Due to its lower density it will layer on top of the Ficoll. Use a stripette for this step and set the pipetboy to a minimum speed. Be careful not to disturb the phase separation between Ficoll and blood when moving the falcon or tilting it back vertically.
3. Centrifuge for 1500 rpm for 25 minutes with break OFF!
4. After centrifugation, you will see a red pellet (with erythrocytes and granulocytes e.g. neutrophils), the Ficoll-paque, a PBMC-ring (peripheral blood mononuclear cells, which includes our monocytes!), and the plasma on top. Remove the plasma, and slowly take off the PBMC ring, again with a strippete and pipetboy in circular motions. Avoid taking the Ficoll with the PBMCs.
5. Wash the PBMCs by transferring them into a fresh 50 ml tube and fill up the falcon with PBS.
6. Centrifuge for 10 minutes at 1500rpm with break ON!
7. For magnetic isolation (positive selection), resuspend the cell pellet in 1ml MACS buffer (PBS + 0.5% BSA, 2mM EDTA).
8. For the kit mentioned above, see manufacturer instructions. We added 75 ul of the CD14 MicroBeads and incubated the cells for 15 minutes on ice.
9. In the meantime, put the LS columns into the magnet and prime them with 3 ml MACS Buffer.
10. Let the labeled cells run through the column, and wash 2x with 5 ml MACS buffer.
11. Since the monocytes are magnetically labeled, they are now stuck inside the column. To remove them from the column, remove the column from the magnet and push them with the provided plunger into a fresh 15 ml tube.
12. Count your cells and centrifuge them for 5 minutes at 1500 rpm.
13. Remove the supernatant and resuspend the monocytes in RPMI containing 10% human serum and 1% penicillin-streptomycin and add 100 ng/ml LPS overnight before use the next day. Primary monocytes can e.g. be seeded at 2x10⁶ cells/ml in a 6-well plate (2–3 ml/well) or at 2x10⁶/well in a 48-well plate.

Caution: All samples should be processed in an appropriate BSL cell culture hood.

Tips

• If you want to separate the plasma from the blood cells, simply centrifuge the whole blood tubes at 2000 rpm for 10 minutes.
• Alternatively, monocytes can be purified by negative selection using e.g. the RosetteSep human monocyte enrichment cocktail before performing the Ficoll density centrifugation. This kit labels all other cells besides the monocytes which can make sense if you e.g. want to stain CD14 with an antibody or want to do functional studies on this receptor afterward.
• When isolating a cell type via positive selection, check which other cell types could also possibly express this surface protein.