THP1 Culture and Differentiation - Revision history

Admin: /* Protocol */

2024-08-21T17:49:38Z

Protocol

Admin: /* Subtype-Specific Macrophage Differentiation */

2024-08-21T14:58:50Z

Subtype-Specific Macrophage Differentiation

← Older revision		Revision as of 14:58, 21 August 2024
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	* 10 mL of RPMI + 10% FBS		* 10 mL of RPMI + 10% FBS
	* 10 uL of 100 ~~ng/mL~~ PMA		* 1.0 uL of 100 uM PMA

	===== Protocol =====		===== Protocol =====

	#Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.		#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 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 of 5 ng/mL).		# 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>.		# Culture for 24 hours at 37C/5% CO<sub>2</sub>.

Admin at 15:13, 7 August 2024

2024-08-07T15:13:33Z

@@ Line 12: / Line 12: @@
 ==== Subtype-Specific Macrophage Differentiation ====
-To be added
 ----

Admin at 19:04, 29 July 2024

2024-07-29T19:04:56Z

← Older revision		Revision as of 19:04, 29 July 2024
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	----		----

	'''Classical Macrophage Differentiation'''		==== '''Classical Macrophage Differentiation''' ====

	# Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.		# 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 ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
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	----		----
	===== Subtype-Specific Macrophage Differentiation =====
			==== Subtype-Specific Macrophage Differentiation ====
	To be added		To be added

	----		----
	===== Dendritic Cell Differentiation =====
			==== Dendritic Cell Differentiation ====
	Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]		Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]

	=====~~= ''iDC Medium'' =~~=====		===== Media Preparation =====

			====== iDC Medium ======
	* 10 mL RPMI + 10% FBS		* 10 mL RPMI + 10% FBS
	* 35 μL 2-mercaptoethanol		* 35 μL 2-mercaptoethanol

Admin at 19:04, 29 July 2024

2024-07-29T19:04:00Z

← Older revision		Revision as of 19:04, 29 July 2024
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	==== Maintenance ====		==== 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.		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.
			----

	'''Classical Macrophage Differentiation'''		'''Classical Macrophage Differentiation'''
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	# Add 10 uL of 100 ng/mL PMA (final concentration of 100 ng/mL), culture for 3-5 days.		# Add 10 uL of 100 ng/mL PMA (final concentration of 100 ng/mL), culture for 3-5 days.

			----
	===== Subtype-Specific Macrophage Differentiation =====		===== Subtype-Specific Macrophage Differentiation =====
	To be added		To be added

			----
	===== Dendritic Cell Differentiation =====		===== Dendritic Cell Differentiation =====
	Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]		Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]

Admin: Created page with "==== 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 10⁶ cells/mL. Do not allow cell density to exceed 1.5 x 10⁶ cells/mL. '''Classical Mac..."

2024-07-29T19:02:53Z

Created page with "==== 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 106 cells/mL. Do not allow cell density to exceed 1.5 x 106 cells/mL. '''Classical Mac..."

New page

==== 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 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 =====
To be added

===== Dendritic Cell Differentiation =====
Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]

====== ''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.

← Older revision		Revision as of 17:49, 21 August 2024
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	====== M0 Differentiation ======		====== M0 Differentiation ======

	# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed ~~PMA~~.		# 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).		# Replace medium with RPMI + 10% FBS (no PMA).
	# Culture an additional 48 hours.		# Culture an additional 48 hours.
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	====== M1 Differentiation ======		====== M1 Differentiation ======

	# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed ~~PMA~~.		# 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.		# Replace medium with RPMI + 10% FBS (no PMA) + 1 μL/mL LPS + 1 μL/mL INFγ.
	# Culture an additional 48-72 hours.		# Culture an additional 48-72 hours.
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	====== M2 Differentiation ======		====== M2 Differentiation ======

	# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed ~~PMA~~.		# 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		# Replace medium with RPMI + 10% FBS (no PMA) + 2 μL/mL IL-4
	# Culture an additional 48-72 hours.		# Culture an additional 48-72 hours.