THP1 Culture and Differentiation
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Maintenance
THP1 cells (ATCC TIB-202) should be maintained in RPMI + 10% FBS, with cells split 1:5 into fresh medium when the cell density reaches 1 x 106 cells/mL. Do not allow cell density to exceed 1.5 x 106 cells/mL.
Classical Macrophage Differentiation
- Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
- Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
- Add sufficient medium to bring the well volume up to 1 mL.
- Add 10 uL of 100 ng/mL PMA (final concentration of 100 ng/mL), culture for 3-5 days.
Subtype-Specific Macrophage Differentiation
Differentiation Medium
- 10 mL of RPMI + 10% FBS
- 1.0 uL of 100 uM PMA
Protocol
- Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
- Add 200 uL of THP1 cells, aiming for 150,000 to 200,000 undifferentiated THP1 cells in each well.
- Add 1 mL of M0 differentiation medium (final PMA concentration at 1.2 mL is 5 ng/mL).
- Culture for 24 hours at 37C/5% CO2.
M0 Differentiation
- After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
- Replace medium with RPMI + 10% FBS (no PMA).
- Culture an additional 48 hours.
M1 Differentiation
- After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
- Replace medium with RPMI + 10% FBS (no PMA) + 1 μL/mL LPS + 1 μL/mL INFγ.
- Culture an additional 48-72 hours.
M2 Differentiation
- After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
- Replace medium with RPMI + 10% FBS (no PMA) + 2 μL/mL IL-4
- Culture an additional 48-72 hours.
Dendritic Cell Differentiation
Protocol from PMID 36674966
Media Preparation
iDC Medium
- 10 mL RPMI + 10% FBS
- 35 μL 2-mercaptoethanol
- 100 μL of 100X antibiotic/antimycotic
- 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
- 10 μL of IL-4 (10 μg/mL stock, 100 ng/mL final)
mDC Medium
- 10 mL RPMI + 10% FBS
- 35 μL 2-mercaptoethanol
- 100 μL of 100X antibiotic/antimycotic
- 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
- 20 μL of IL-4 (10 μg/mL stock, 200 ng/mL final)
- 20 ng/mL TNFα
- 200 ng/mL ionomycuin
iDC Procedure
- Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
- Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
- Add 1 mL of iDC medium to each well, incubate 3 days.
- Replace medium and incubate an additional 2 days.
mDC Procedure
Starting with iDCs from above
- Replace medium with mDC medium.
- Incubate 2 days.