For my project I have to pretreat RAW 264.7 macrophages with adenosine or 2-chloroadenosine for 1h and 24h, then followed with LPS (1ug/mL) treatment for another 24h. However, I'm not sure whether I need to wash my cells prior to adding medium containing LPS.
Please see attached journal article:
https://jpet.aspetjournals.org/content/316/1/71
Hello Armando Huang,
Yes, you have to wash
As per your experimental protocol, your objective is to pre-treat the RAW 264.7 macrophages with adenosine or 2-chloroadenosine for 1h and 24h in order to inhance the anti-inflamtory capacity of cells and then then you are transferring the cells in a medium containing LPS (1ug/mL) for another 24h.
The washing of cells with appropriate plain medium is important to remove the anti-inflamatory compounds from the culture medium and then only you can analyze the exact effects of LPS (1ug/mL) or preventive effects of inflammatory compounds.
Otherwise, it will be partly like a co-incubation of both drugs (anti-inflamatory and inflamatory effects) and may cause even drug-drug interaction in the culture medium itself (this happens with certain drugs). Under this condition, you may not be in position to scientifically report the effects of any particular concentration of any drug on the cells of your interest under in vitro culture conditions.
Hence, wash the cells at least three times in order to remove the trace of adenosine or 2-chloroadenosine present in the culture medium and then expose with LPS (1ug/mL) for another 24h.
Good luck
- Badges
- Science method