TITLE: Culturing THP-1 Cells
AUTHORS: Teesha Luehr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">THP-1 cells are a human monocyte suspension cell line from peripheral blood of a 1 year old infant who had acute mnocytic leukemia. </div></div>

[STEPS]
?. [Preparing Media]
The base medium for this cell line is RMPI-1640
?. [Preparing Media]
Reguired supplements:
[L-glutamine]
[Fetal Bovine Serum]
Most catalog numbers of RMPI-1640 contain L-glutamine, however, some do not. Ensure that it is in the media before using it for culturing.
?. [Preparing Media]
Optional Supplements:
[PenStrep]
[2-mercaptoethanol]
PenStrep is not required for THP-1 culturing, however, if you are having issues with bacterial contamination, it can be used at 1X.
2-mercaptoethanol is stated as a required component for complete RPMI-1640 medium, however, in our laboratory it is not standard practice to add it.
?. [Cell Storage]
Always store cells in liquid nitrogen. This is for both the original tube of cells from ATCC and any passages afterwards.
?. [Preparation of Materials & Reagents ]
Place the media bottle in the at least
[water bath]
[prior to using]
?. [Preparation of Materials & Reagents ]
Thaw cells at for  or for
[(room temperature)]
[in a water bath]
?. [Working from fozen cells]
As soon as the cells are thawed, transfer the cells to a and add
[conical tube]
[of complete media]
Cells are stored with 5% DMSO, which can lyse cells if they are left for too long.
?. [Working from fozen cells]
Pellet cells for
[at 500g]
?. [Working from fozen cells]
Discard supernatant
?. [Working from fozen cells]
Resuspend cells by pipetting up and down 5X in
[complete media]
?. [Working from fozen cells]
Sanitize all items going into the Biological Safety Cabinet with 70% ethanol
?. [Working from fozen cells]
Transfer cells + media to a T-25 flask
?. [Incubation]
Incubate cells at
[and 5% CO2 and 80% humidity]
?. [Feeding and Splitting]
THP-1 cells replicate after ~26 hours. In practice, it takes 2 days for a true doubling.
?. [Feeding and Splitting]
Once cells have doubled OR when media has begun to change colour, it is time to add media, split cells into new flasks, or to spin down to remove all media
?. [Feeding and Splitting]
If concerned about cell concentration, perform a cell count
?. [Feeding and Splitting]
Double the total media volume with new complete media
?. [Feeding and Splitting]
If concerned about cell concentration, perform a cell count
?. [Feeding and Splitting]
Carefully mix the new media in by rocking the flask back and forth
?. [Feeding and Splitting]
Once the volume limit is reached for the T-25 flask it is time to move to a T-175 flask
?. [Feeding and Splitting]
Remove all cell + media from the T-25 flask and transfer them to the T-175 flask
This has to be done with a  because the larger volume pipettes do not fit in the T-25.
?. [Feeding and Splitting]
Double the total media volume with new complete media
?. [Feeding and Splitting]
If cells are appearing unhappy, it may be beneficial to remove all of the current media and dead cells and replenish with new complete media
?. [Feeding and Splitting]
Transfer all cell + media to 50mL conical tubes
?. [Feeding and Splitting]
Pellet cells for
[at 500g]
?. [Feeding and Splitting]
Discard supernatant
?. [Feeding and Splitting]
Resuspend cells by pipetting up and down 5X in new complete media.
Unless cells were at a drastically low cell concentration, resuspend cells in the same volume of media they were in before pelleting.
?. [Feeding and Splitting]
Transfer cells to a new T-flask
?. [Feeding and Splitting]
Place the flask back in the incubator
?. [Feeding and Splitting]
Place all flasks in the incubator
?. [Feeding and Splitting]
Place flask into the incubator