View source for THP1 Culture and Differentiation

==== Maintenance ====
THP1 cells ([https://www.atcc.org/products/tib-202?matchtype=&network=x&device=c&adposition=&keyword=&gad_source=1&gclid=Cj0KCQjw-5y1BhC-ARIsAAM_oKlZTKs5zplX22cd1pVZB7E0-Cq7fDL5pcruuijrK0PqV9a-5LG5l24aAoEqEALw_wcB 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 10<sup>6</sup> cells/mL. Do not allow cell density to exceed 1.5 x 10<sup>6</sup> cells/mL. 
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==== '''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.

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==== 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% CO<sub>2</sub>.

====== 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.

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==== Dendritic Cell Differentiation ====
Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ 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.