Anti-inflammatory tumor associated macrophages are linked to progression and poor prognosis in cancer. This protocol is a guide to differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. Currently there is no established model for anti-inflammatory THP-1 polarization.
This protocol uses adequate resting periods and a prolonged polarization process to create a distinct M2-like macrophage subtype. Tumor development, for example, in colon cancer and tissue remodeling, are controlled by anti-inflammatory macrophages. Among studies investigating their function, standardized creation of a distinct M2-like subtype ensures reproducible findings.
Begin by setting a timer for 150 seconds. Remove the frozen vial containing the THP-1 cell line from the liquid nitrogen and thaw the vial immediately at 37 degrees Celsius in a clean water bath, starting the timer as soon as the vial is put into the water bath. Loosen the cap to release the pressure built up due to the thawing process, ensuring that the tube opening does not contact the water to avoid contamination.
Thaw the cell suspension until an ice chip about four millimeters in size is left within the vial and proceed to the next step immediately. Transfer the liquid phase of the cell suspension to a 15 milliliter tube containing nine milliliters of warm growth media. Then transfer one milliliter of this warm medium cell suspension into the THP-1 vial and back to the 15 milliliter tube to melt the remaining ice chip, and flush the vial to ensure no cells are left behind.
Mix the suspension gently by pipetting it up and down with a one milliliter pipette. Remove approximately 10 microliters of the sample to count the cells for viability using trypan blue. The cells with clear cytoplasm are viable, a blue cytoplasm indicates cell death.
While counting, spin down the warm cell suspension at 200 times G for seven minutes at 37 degrees Celsius. Remove the supernatant completely and resuspend the cells in growth medium to achieve a cell density of 500, 000 cells per milliliter. Mix the suspension gently and transfer 22 milliliters into T75 cell culture flasks each.
Store the flasks upright in an incubator at 37 degrees Celsius with a 5%carbon dioxide concentration and exchange the growth media every three to four days. Prepare the cell suspension to seed the cells at a density of 300, 000 per milliliter per well into 24-well cell culture plates. Mix the medium gently and a prepare aliquots of 26 milliliters in 50 milliliter tubes.
Transfer one milliliter of the cell-containing medium into each well of a 24-well plate. Mix the media gently by pipetting it up and down between transfers. Keep the freshly prepared working solution of PMA on ice and add 100 nanograms of PMA per well.
Incubate the plates in the incubator without any further treatment for 72 hours. After 72 hours, remove the growth medium and replace it with one milliliter of fresh growth medium, ensuring not to touch the bottom of the wells with pipette tips. Let the cells rest for another 96 hours in the incubator.
Remove the growth medium and replace it with one milliliter of fresh growth medium. Add 20 nanograms of interleukin 4 and 20 nanograms of interleukin 13 per well. Then let the cells rest for 48 hours in the incubator.
Repeat the whole process after 48 hours and let the cells rest for another 48 hours in the incubator. Remove the growth medium and replace it with one milliliter of fresh growth medium. Then let the cells rest for 48 hours in the incubator.
Remove the warm cell medium from each well and replace it with a one milliliter mixture of ice cold PBS without calcium and magnesium and 5%FBS. Then immediately place the cell plate on ice for 45 minutes. After 45 minutes on ice, scrape off the cells using mini cell scrapers.
Gently transfer the detached macrophages to cold PBS and 5%FBS in a 15 milliliter tube kept on ice. The macrophages derived from the polarization method show the expression of CD14, as well as CD11b markers. The monocyte and macrophage markers CD14 and CD11b were expressed in 70.9%and 74.7%of cells, respectively.
Cells showed low surface levels of the M1-like marker CD80 in 0.2%of cells, and high surface levels of the M2-like marker CD206 in 62.6%of cells. As THP-1 cells in medium tend to sink to the bottom of a vial and are easy to activate through mechanical stress, gentle mixing and handling of the cells is important. M2-like macrophages are reproducibly created, and have been characterized using QR-TPCR, ELISA, and flow cytometry.
This is the basis for investigating anti-inflammatory mechanisms in cancer development or tissue remodeling.
