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.

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 * 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