THP-1 Cell Differentation

Macrophages are important immune effector cells and play an important role in innate and adaptive immune responses. THP-1 cells are usually induced to differentiate into macrophages with PMA.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. THP-1 monocytes cultivated in RPMI 10% FBS Pen/Strep in a humidified incubator at 34℃ 5% CO₂. Centrifuge at 200 x g at room temperature for 8 min, discard culture media and resuspend pelleted cells in a volume sufficient for counting.

2. Mix 10 µL of the resuspended cells with 10 µL of Trypan Blue. Count and evaluate cell viability and calculate the concentration of cell per mL. Dilute cells/mL according to the preferred plate for 24 well plate 1×10⁶ cells/well, 6 well plate 5×10⁶ cells/well.

3. Add PMA: Final concentration for THP-1 differentiation: 30 ng/mL diluted in RPMI 10% FBS. Store for 72 h at the incubator 34 ℃ 5% CO₂.

4. Wash cells 3 times with PBS 1X to remove residual PMA.

5. Incubate cells with fresh RPMI 10% FBS medium for further 72 h 34 ℃ 5% CO₂.

6. Proceed to the assay with fully differentiated THP-1 macrophages.

1. If there are cell debris during the culture process, wait until the cells grow up and then remove them by low-speed centrifugation.

2. When a small amount of cell aggregation occurs, we can wait for the THP-1 cell density to expand and improve the situation. However, when severe aggregation occurs, we can increase the amount of serum (FBS) or blow the cells appropriately.

3. THP-1 cells may appear adherent after passage. If there is slight adhesion, you can gently blow to collect the cells or discard the adherent cells. If there is a lot of adhesion, you can check whether the culture conditions are suitable.