Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.
HIV infection causes immune dysfunction even in individuals undergoing antiretroviral therapy with no detectable virus. This method allows for the study of monocyte function which are key regulators of the immune response. We have optimized this technique for the isolation, culture, and transfection of human primary monocytes.
We use this method to understand the molecular mechanisms of immune dysfunction in HIV infected individuals, but it can be applied to any other immune studies involving monocytes. If this is your first time working with human blood, remember to follow the proper biosafety protocols and to treat all of the samples as possibly infectious material. After collecting 40 milliliters of fresh human whole blood in four 10 milliliter EDTA vacuum tubes, use sterile technique to transfer all of the blood into a single 50 milliliter conical propylene tube in a biosafety cabinet.
Following the manufacturer's instructions from a selected human monocyte isolation kit, add two milliliters of monocyte isolation cocktail from the kit to the tube of blood and vortex the magnetic beads from the kit for 30 seconds. Add two milliliters of beads to the blood and use a 25 milliliter serological pipette to carefully mix the beads with the blood. After five minutes at room temperature, split the blood equally between four 50 milliliter tubes and add 30 milliliters of sterile PBS supplemented with one millimolar EDTA to each tube.
Mix again with a plastic 25 milliliter serological pipette and place the tubes in magnetic holders. After 10 minutes, use a pipette to draw up the contents from the center of each tube taking care not to aspirate more than one milliliter of red blood cells and dispense the tube contents into one of four new 50 milliliter tubes. Add 500 additional microliters of the vortexed magnetic beads to each tube and gently mix the cell solution.
Place the tubes back into the magnetic holders for five minutes before carefully transferring the contents from the center of each tube into one of four new 50 milliliter tubes. When all of the cell suspensions have been transferred, place each new 50 milliliter tube into the magnet holders for five minutes before carefully transferring the tube contents into a third set of four new 50 milliliter tubes. Then collect the cells by centrifugation and resuspend all four cell pellets in a total of 10 milliliters of sterile PBS for counting.
After counting, resuspend the isolated monocytes at a one times 10 to the sixth cells per milliliter of 37 degrees Celsius serum-free RPMI 1640 medium supplemented with antibiotics and plate one milliliter of resuspended cells into 35 millimeter plates in a biosafety cabinet. Then place the plates in the cell culture incubator for 30 to 60 minutes to allow the cells to adhere to the well bottoms. To prime for macrophages with an M1-like phenotype, add 100 microliters of fetal bovine serum containing 25 nanograms per milliliter of GM-CSF to each plate.
To prime for macrophages with an M2-like phenotype, add 100 microliters of fetal bovine containing 50 nanograms per milliliter of M-CSF to each plate. For monocyte transfection with microRNA mimics, inhibitors, or a small interfering RNA, following the manufacturer's protocol for the transfection kit, dilute the selected RNAs in the buffer to a final concentration of 1.83 micromolar in 10 microliters of diluted mimic or inhibitor per transfection of one times 10 to the fifth cells volume. To prepare the transfection reagent, add one microliter of the provided polymer to a 1.5 milliliter microcentrifuge tube and immediately add 90 microliters of the kit-provided buffer to obtain a total of 91 microliters of reagent per transfection.
Vortex the regent for three to five seconds and pipette 90 microliters of the transfection solution into the tube containing 10 microliters of diluted RNA. After gentle mixing, incubate the solution for 15 minutes at room temperature before adding 100 microliters of transfection complex to one well of cells. After four hours at 37 degrees Celsius, replace the medium with three milliliters of complete medium containing the appropriate growth factor.
On day six of culture, replace the culture supernatants from the M1 cultures with three milliliters of medium supplemented with fetal bovine serum, antibiotics, lipopolysaccharide, and interferon gamma and replace the supernatants from the M2 cultures with medium supplemented with fetal bovine serum, antibiotics, M-CSF, and interleukin-4. After 24 hours of culture, wash each plate of activated macrophages two times with fresh PBS per wash. For RNA and protein analyses, lyse the attached cells directly in the plates to allow collection of the released cell contents for their analysis.
For flow cytometric analysis, detach the cells with two millimolar EDTA in PBS for 10 minutes at 37 degrees Celsius before gently scraping the cells from the plate bottoms to allow their collection into 1.5 milliliter microcentrifuge tubes for their analysis. Here representative histograms of M1-activated control and patient-derived cells with increased levels of CD80 and CD83 and M2-activated cells with increased levels of CD163 and CD209 as compared to non-activated T0 cells are shown. Interestingly, CD80 and CD83 appeared to be more highly expressed in control-derived cells compared to HIV-derived cells.
The transfection of freshly collected monocytes with a scrambled near-infrared labeled microRNA results in a greater than 90%efficiency of transfection as determined by flow cytometry and confocal microscopy. In addition, transfection does not significantly reduce the viability of patient-derived or control cells regardless of the stage of cell maturation or the transfection conditions. Transfection results in an effective downregulation of the target gene upon transfection with the specific small interfering RNA at both day one and day four post-isolation.
Further, cells transfected with microRNA mimic demonstrate a 48 to 72-fold increase in microRNA expression over untransfected cells while all of the transfection controls show no appreciable changes. The number of cells plated is critical for survival. We recommend one million cells per 35 millimeter dish.
Plating in serum-free medium will also greatly improve the cell attachment. Cells obtained with this method can be treated with cytokines or growth factors to study differential responses in patients of interest and healthy controls.
