Can you share your protocol for preparing acetone-precipitated cell media from TPA-differentiated THP-1 cells treated with LPS?

Our protocol is as follows:

REAGENTS:
TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174
Lipopolysaccharides (LPS) #14011

DAY 1:

1. Seed THP-1 cells at approximately 8 X 10^5 cells/mL in 15 cm plates with 20 ml RPMI +10% FBS + 0.05 mM 2-mercaptoethanol. (We use plates instead of flasks since the cells adhere to the plate after treatment with TPA).
2. Treat plates overnight with 80nM TPA.

DAY 2:

1. Wash treated plates with 10 mL 1x PBS, drain, and add fresh media (RPMI +10% FBS + 0.05 mM 2-mercaptoethanol).
2. Allow cells to rest overnight.

DAY 3:

1. Remove media from plates.
2. Add 15 mL serum-free RPMI to plates.
3. Treat plates with LPS at 1ug/mL for 8 hours.
4. Collect media after 8 hours at centrifuge at 1500 - 2000 RCF for 5 minutes.
5. While the media is spinning, wash the cells from the TPA/LPS treated plates with 1X PBS and lyse in 1X SDS/DTT (we use 500uL - 1mL 1X SDS+DTT per plate). Sonicate lysate on ice and boil at 95C for 5 minutes. Store at -80C until ready to use. 
6. Collect media from centrifugation and transfer to a new 50mL conical. You may or may not observe a pellet after spinning – discard the pellet if there is one. 
7. Add cold acetone to the media (1 part media: 2 parts acetone). Place at -80C overnight.

DAY 4:

1. Thaw acetone precipitated media conical tube on ice.
2. Centrifuge between 6800 RCF and 14,000 RCF (use max possible depending on centrifuge capability and rating of the conical tubes) at 4C for 10 minutes.
3. Remove acetone supernatant (collect as hazardous waste) and add the desired volume of 1X SDS +DTT to the pellet (We use 350 uL 1X SDS +DTT per initial 15 cm plate of cells used to generate the sample). 
4. Do not sonicate.
5. Boil at 95C for 5 minutes.
6. Store at -80C until ready for use. 

Last updated: September 12, 2024