Moghe:THP-1

THP-1 Procedures

Subculture: It is recommended to subculture the cells at 3.5x105 viable cells/mL. Feed the cells every 3-4 days and passage cells every 5-7 days (cycle is cell dependent)

Thaw:

1. Thaw the cells in a 37ºC water bath by dipping the vial in while keeping the cap above water
2. Remove the vial as soon as it becomes mostly liquid and wipe down the sides with 70% ethanol
3. Transfer the contents to a centrifuge tube and add 5mL of fresh room temperature FBS RPMI and place into the appropriate number of T25 flasks with 5mL FBS RPMI for 1x106 in each flask
4. Place flasks in the 37ºC, 5% CO2 incubator with caps loose
5. The next day transfer the contents to a centrifuge tube and add 5mL of fresh warm FBS RPMI and spin down for 5 minutes at 1000 rpm
6. Resuspend the pellet into the appropriate number of T25 flasks with 5mL FBS RPMI for each flask
7. Place flasks in the 37ºC, 5% CO2 incubator with caps loose

Feeding Cells:

1. Warm up the appropriate amount of fresh FBS RPMI, 2-3mL per T25 flask and 5-7mL per T75 flask, in a 37ºC water bath
2. Add the fresh FBS RPMI to the existing flask
3. Place flasks in the 37ºC, 5% CO2 incubator with caps loose

Passaging Cells:

1. Transfer the contents of flasks into 15mL centrifuge tubes and spin down for 5 minutes at 1000 rpm
2. While the cells are spinning, warm up the appropriate amount of fresh FBS RPMI, 5mL per T25 flask and 15mL per T75 flask, in a 37ºC water bath
3. Resuspend the cells into the appropriate volume of media to correspond to the number of T25 flasks with 5mL medium each (or 15mL for T75)
4. Place flasks in the 37ºC, 5% CO2 incubator with caps loosened

Differentiating cells:

1. Transfer the contents of flasks into 15mL centrifuge tubes and spin down for 5 minutes at 1000 rpm.
2. While the cells are spinning, prepare and warm up the appropriate amount of differentiation medium to fill the plates required for the experiment, in a 37 ºC water bath.

• Differentiation medium - 15uL of TPA medium + 15mL medium = 16x10-9 M

1. Resuspend the pellet in 3-4mL of warm differentiation medium and count the cells
2. Seed at 30,000 viable cells/well for a 96 well plate
3. Place flasks in the 37ºC, 5% CO2 incubator
4. After 14hr incubation, carefully remove the differentiation medium from the wells and replace with fresh warm FBS RPMI
5. Replace flasks in the 37ºC, 5% CO2 incubator and allow an additional 58hr incubation for a total of 72hr before performing any experiments

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