In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Isolation of primary human monocytes by immunomagnetic negative selection

1. Collect 40 mL of fresh, human whole blood (from either an HIV⁺ patient or healthy control) in four 10 mL ethylenediaminetetraacetic acid (EDTA) vacuum tubes (10 mL of blood per tube). Using sterile techniques under a biosafety cabinet, transfer all 40 mL of blood into one 50 mL conical propylene tube.
2. Following the manufacturer's protocol for the selected human monocyte isolation kit (Table of Materials), add 2 mL of monocyte isolation cocktail, provided in the kit, to the tube of blood. Vortex magnetic beads, also provided in the kit, for 30 s, and add 2 mL to the tube of blood.
   1. If less than 40 mL of blood is available, scale down the reagents added. To mix the solution, pipette up and down with a plastic 25 mL serological pipette and incubate for 5 min at room temperature (RT).
3. Separate the blood mixture equally into four 50 mL tubes and add 30 mL of sterile phosphate-buffered saline (PBS) containing 1 mM EDTA to each tube. Mix by pipetting up and down with a plastic 25 mL serological pipette.
4. Place the tubes in magnet holders for 10 min to remove the antibody-conjugated magnetic beads. Use four magnet holders simultaneously, one for each tube, to allow consistent incubation and isolation times for each blood sample.
5. Draw up the contents from the center of each tube, using a pipette, while they are still in the magnet holders. Be careful not to draw up red blood cells (no more than 10% of the 10 mL starting volume), and place the contents into one of four new 50 mL tubes.
6. Add 500 µL of vortexed magnetic beads to each 50 mL tube. Pipette up and down with a 25 mL pipette and incubate at RT for 5 min. Then, place the tubes into magnet holders for 5 min.
7. Carefully transfer the contents from the center of each tube while still in magnet holders into one of four new 50 mL tubes. Directly place each new 50 mL tube in the magnet holders for 5 min.
8. Carefully transfer contents from the center of each tube into one of four new 50 mL tubes. Spin all new 50 mL tubes at 300 x g for 5 min. Aspirate the supernatant and resuspend all four cell pellets in a total of 10 mL of sterile PBS.
9. Count cells by trypan blue exclusion using a hemocytometer.
   NOTE: 8-20 x 10⁶ cells are generally obtained from 40 mL of whole blood.

2. Culturing of primary human monocytes

1. Using a 37 °C water bath, warm serum-free RPMI 1640 media supplemented with 1% penicillin-streptomycin (pen/strep), and (while continuing to use sterile techniques under a biosafety cabinet) resuspend the isolated monocytes in this media at a concentration of 1 x 10⁶ cells/mL.
2. Add 1 mL of resuspended cells to each well of a 6-well plate or into a 35 mm dish (the final number of cells should be 1 x 10⁶ cells/plate), and place in a 37 °C incubator with 5% CO₂. Wait for 0.5-1.0 h for cells to adhere.
3. Using a 37 °C water bath, warm heat-inactivated (HI) fetal bovine serum (FBS). Add 100 µL (10% final concentration) of FBS to each plate. Add growth factors to the cells to promote macrophage differentiation.
   1. Prime macrophages for an M1-like phenotype by adding 25 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) to media. Prime macrophages for an M2-like phenotype by adding 50 ng/mL of macrophage colony-stimulating factor (M-CSF) to media.
      NOTE: Both GM-CSF and M-CSF allow monocyte differentiation to a general macrophage phenotype (M0) while priming cells for M1 or M2, respectively.