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

  1. Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
  2. Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
  3. Add sufficient medium to bring the well volume up to 1 mL.
  4. 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
  1. Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
  2. Add 200 uL of THP1 cells, aiming for 150,000 to 200,000 undifferentiated THP1 cells in each well.
  3. Add 1 mL of M0 differentiation medium (final PMA concentration at 1.2 mL is 5 ng/mL).
  4. Culture for 24 hours at 37C/5% CO2.
M0 Differentiation
  1. After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
  2. Replace medium with RPMI + 10% FBS (no PMA).
  3. Culture an additional 48 hours.
M1 Differentiation
  1. After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
  2. Replace medium with RPMI + 10% FBS (no PMA) + 1 μL/mL LPS + 1 μL/mL INFγ.
  3. Culture an additional 48-72 hours.
M2 Differentiation
  1. After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
  2. Replace medium with RPMI + 10% FBS (no PMA) + 2 μL/mL IL-4
  3. 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
  1. Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
  2. Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
  3. Add 1 mL of iDC medium to each well, incubate 3 days.
  4. Replace medium and incubate an additional 2 days.
mDC Procedure

Starting with iDCs from above

  1. Replace medium with mDC medium.
  2. Incubate 2 days.