The overall goal of the following experiment is to isolate monocytes from human peripheral blood and to differentiate them toward different macrophage polarization types. This is achieved by first isolating human peripheral blood mononuclear cells by density gradient centrifugation. As a second step negative bead isolation is performed to obtain pure CD 14 positive monocytes.
Next monocytes are cultured under specific conditions in order to induce specifically polarized macrophages. Ultimately, the differential expression of macrophage polarization markers in response to their culture conditions can be determined by quantitative PCR. This method can help to dissect the differentiation of macrophages towards different polarization types.
A feature that is extremely important in inflammatory diseases such as atherosclerosis. After drawing 30 milliliters of whole blood from the forearm vein from a volunteer, split the blood evenly into two non polystyrene 50 milliliter conical tubes. Then dilute the blood in each tube with 10 milliliters of PBS.
Next, add 25 milliliters of histo pack to the 25 milliliters of whole blood and PBS in each tube, taking care that the histo pack and blood do not mix. Then centrifuge the tubes for 30 minutes at 400 Gs and room temperature with no break. After the spin first aspirate the plasma layer and then transfer the opaque interface layer into a fresh 50 milliliter tube.
Wash the cells from the interface in 20 milliliters of PBS plus CDTA for five minutes at 250 Gs and four degrees Celsius. Then after discarding the supernatant resus, suspend the pellet in 10 milliliters of PBS plus CDTA and mix the cells well by vortexing for 10 seconds. Now dilute 200 microliters of the cell suspension in 300 microliters of PBS plus EDTA, and 500 microliters of triam blue.
To achieve about 200 to 500 cells per 10 squares and a hemo cytometer begin the negative isolation by spinning down the cells three times for 10 minutes at 120 Gs After the second wash, we suspend the pellet in nine milliliters of sterile water for three seconds. Then add one milliliter of 10 XPBS after washing and counting the cells one more time, dilute them in easy set buffer at five times 10 of the seven cells per milliliter. Now incubate the cells for 10 minutes at four degrees Celsius in 50 microliters per milliliter of monocyte enrichment cocktail.
During the last minute of the incubation vortex the cept monocyte enrichment beads for 30 seconds. Then add 50 microliters per milliliter of the beads to the cells and incubate the bead in cell suspension for another five minutes. After the second incubation, bring the cell solution volume up to 2.5 milliliters.
With more eep buffer, the solution will now appear a brownish color. Next, place the tube into an eep magnet for two and a half minutes at room temperature. After the bead bound cells have had a chance to stick, pour the buffer containing the non-binding monocytes into a fresh sterile tube.
The solution will now appear a whitish color after washing the unbound cells once with PBS plus EDTA, transfer the cells into a multi-well plate, then add one milliliter of culture media per one times 10 to the six cells to the culture plate. Finally, culture the cells under the experimental conditions of interest for six days to induce macrophage differentiation. Replace half of the media after three days with fresh media after six days.
Harvest the cells for the desired experimental analysis. Using the protocol described above about 25 times 10 of the six monocytes are routinely obtained from 100 milliliters of blood monocyte purity as determined by flow cytometric staining for CD 14 is routinely greater than 95%when first added to the culture. The monocytes are initially round shaped and float within a few hours.
However they adhere giving them a fried egg or spindle like appearance after six days. In culture with MCSF alone or with the addition of oxidized LDL for the last 24 hours or roughly 80%of the cells not exposed to oxidized LDL are viable as determined by propidium iodide. Staining treatment with oxidized LDL on the other hand results in about 70%cell viability after six days of culture.
With MCSF flow, CYTOMETRIC analysis demonstrates that while the cells keep expressing the leukocyte marker CD 45 ro, they downregulate the monocyte marker CD 14. Depending on the specific conditions that may induce macrophage polarization during cell culture, additional markers are necessary to warrant successful polarization. Specific conditions may induce macrophage polarization.
For example, M1 polarized macrophages express typical markers like IL six and TNF alpha, whereas M two polarized macrophages express CD 36 and CD 2 0 6, macrophages can further be studied in functional experiments. For example, the uptake of oxidized LDL can be studied using fluorescently labeled LDL. In this experiment, the cells were exposed to 10 micrograms per milliliter of DII labeled oxidized LDL for four hours, and the nuclei re stain with dpi.
The histogram demonstrates the flow cytometric measurement of the DII oxidized LDL uptake by MCSF induced macrophages with the solid gray histogram representing the untreated control cells and the black open histogram. Representing the oxidized LDL treated macrophages After its development. This technique enables researchers to study differentiation and heterogeneity of human macrophages.
It thereby enables them to study inflammatory diseases such as atherosclerosis to identify novel disease mechanisms and novel therapeutic targets.
