Moghe:THP-1: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Pratik Shah (talk | contribs) No edit summary |
Pratik Shah (talk | contribs) No edit summary |
||
Line 1: | Line 1: | ||
THP-1 Procedures | ==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) | 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: | ===Thaw:=== | ||
#Thaw the cells in a 37ºC water bath by dipping the vial in while keeping the cap above water | |||
#Remove the vial as soon as it becomes mostly liquid and wipe down the sides with 70% ethanol | |||
#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 | |||
#Place flasks in the 37ºC, 5% CO2 incubator with caps loose | |||
#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 | |||
#Resuspend the pellet into the appropriate number of T25 flasks with 5mL FBS RPMI for each flask | |||
#Place flasks in the 37ºC, 5% CO2 incubator with caps loose | |||
Feeding Cells: | ===Feeding Cells:=== | ||
#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 | |||
#Add the fresh FBS RPMI to the existing flask | |||
#Place flasks in the 37ºC, 5% CO2 incubator with caps loose | |||
Passaging Cells: | ===Passaging Cells:=== | ||
#Transfer the contents of flasks into 15mL centrifuge tubes and spin down for 5 minutes at 1000 rpm | |||
#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 | |||
#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) | |||
#Place flasks in the 37ºC, 5% CO2 incubator with caps loosened | |||
Differentiating cells: | ===Differentiating cells:=== | ||
#Transfer the contents of flasks into 15mL centrifuge tubes and spin down for 5 minutes at 1000 rpm. | |||
#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 | *Differentiation medium - 15uL of TPA medium + 15mL medium = 16x10-9 M | ||
#Resuspend the pellet in 3-4mL of warm differentiation medium and count the cells | |||
#Seed at 30,000 viable cells/well for a 96 well plate | |||
#Place flasks in the 37ºC, 5% CO2 incubator | |||
#After 14hr incubation, carefully remove the differentiation medium from the wells and replace with fresh warm FBS RPMI | |||
#Replace flasks in the 37ºC, 5% CO2 incubator and allow an additional 58hr incubation for a total of 72hr before performing any experiments | |||
<nowiki><nowiki>Insert non-formatted text here</nowiki><nowiki><nowiki>Insert non-formatted text here</nowiki><nowiki><nowiki>Insert non-formatted text here</nowiki></nowiki></nowiki></nowiki> | <nowiki><nowiki>Insert non-formatted text here</nowiki><nowiki><nowiki>Insert non-formatted text here</nowiki><nowiki><nowiki>Insert non-formatted text here</nowiki></nowiki></nowiki></nowiki> |
Latest revision as of 14:43, 25 October 2010
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:
- Thaw the cells in a 37ºC water bath by dipping the vial in while keeping the cap above water
- Remove the vial as soon as it becomes mostly liquid and wipe down the sides with 70% ethanol
- 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
- Place flasks in the 37ºC, 5% CO2 incubator with caps loose
- 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
- Resuspend the pellet into the appropriate number of T25 flasks with 5mL FBS RPMI for each flask
- Place flasks in the 37ºC, 5% CO2 incubator with caps loose
Feeding Cells:
- 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
- Add the fresh FBS RPMI to the existing flask
- Place flasks in the 37ºC, 5% CO2 incubator with caps loose
Passaging Cells:
- Transfer the contents of flasks into 15mL centrifuge tubes and spin down for 5 minutes at 1000 rpm
- 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
- 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)
- Place flasks in the 37ºC, 5% CO2 incubator with caps loosened
Differentiating cells:
- Transfer the contents of flasks into 15mL centrifuge tubes and spin down for 5 minutes at 1000 rpm.
- 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
- Resuspend the pellet in 3-4mL of warm differentiation medium and count the cells
- Seed at 30,000 viable cells/well for a 96 well plate
- Place flasks in the 37ºC, 5% CO2 incubator
- After 14hr incubation, carefully remove the differentiation medium from the wells and replace with fresh warm FBS RPMI
- Replace flasks in the 37ºC, 5% CO2 incubator and allow an additional 58hr incubation for a total of 72hr before performing any experiments
<nowiki>Insert non-formatted text here<nowiki>Insert non-formatted text here<nowiki>Insert non-formatted text here</nowiki></nowiki></nowiki>