THP1 Culture and Differentiation

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.

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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.

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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.

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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.