Could anyone provide me a protocol for endotoxin determination of medium and buffer for RAW 264.7 macrophages? Thank you very much!
A typical test for Endotoxin-levels is the "LImulus Amebocyte assay, some commercial versions you can find here: http://www.genscript.com/endotoxin_assay_kits.html or just google the keywords "Endotoxin assay" Not sure, how sensitive Raw cells are actually. However, typically if you want to investigate LPS response, you need to make sure everything you keep the cells in is endotoxin-free. Otherwise the cells get de-sensitized (personal experience with MonoMac 6 cells (Monocyte cell line, makes TNF alpha upon LPS stimulation, works very nicely, if Raw don't work for you, you could try these instead). Also critical: do not use glass pipettes! Plasticware only! Maybe sounds overprecautious, but is necessary, unfortunately. Glassware is a notorious source for LPS trace amounts and can make the outcome of your experiments unpredictable (PBS in glass bottles is another typical issue). Also make sure your batch of FCS is tested low endotoxin. There is a high variation, don't just rely on sales reps, they will always tell you "We have the best". when you have kept your cells under low enotoxin for a few passages, you're ready to go. You can try without, but for me this made results a LOT more reproducible.... ;-)
More questions? just post here. Good luck!
Thomas :-)
Dear Alexander
A note regarding FCS. As mentioned above be sure the FCS you use is low endotoxin, get the Data sheet for the specific batch from the vendor and order plenty for your entire study.
good luck
Jesper
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