Hi Scientist Solutions community,
I have been having problems inducing thioglycollate-induced peritonitis in mice. The purpose of the experiment is to observe migration defects in peripheral macrophages. Unfortunately, I have had inconsistencies in my FACS data (staining for F4/80) that haven't gone away. I have gone from observing a 12-20% increase in infiltrating macrophages (F4/80 dim) to 2-6%. I was wondering if anyone could help me fix the protocol so a greater difference can be observed.
1) Thioglycollate used: 3 mL of 3% thioglycollate (made in Saline buffer)
(From Sigma Aldrich. NOT Brewer's. CAT#: 70157 )
**I don't autoclave it. I only make 15 mL at a time, and under a biosafety cabinet. I make the solution the day before, and have been leaving it at 4C overnight.
Questions: Should I store the thioglycollate in the fridge overnight or just leave it at room temperature? And will autoclaving/sterilizing the solution really make a difference?
I have read that 'aged' thioglycollate (autoclaved and fridged for at least a week) helps a lot. I was wondering if anyone knew the mechanism of thioglycollate for peritonitis. In any case, the reason I haven't tried to age thioglycollate is because I'm wary of the idea that 2 week old thioglycollate would induce a greater response than 1 week old thioglycollate. Which is to say unless I'm making a batch a week, my results will end up with a large range...
2) Wait 24 hours (sometimes an hour more, but never less)
I know macrophages take up to 2-3 days to fully migrate, so I was wondering if I should wait longer (the paper I based my protocol on used 3 mL 3% thio for 24 hours. The peritonitis was not the focus of their paper, though, and so I don't have many figures I can refer to. I'm plotting my FACS as SSC-A vs F4/80).
3) Remove cells with 10 mL cold PBS using a procedure similar to http://www.jove.com/index/details.stp?ID=1488">this
**There has been some blood contamination, as I am still new to the procedure. The times I have gotten only 2-6% difference in macrophage population are more recent, where I am getting very little blood contamination now. It seems I observe blood entry when I cut open the mouse to pipette out the remainder of the PBS, so I have actually skipped that step (total collection is about 8-9 mL, though I inject 10 mL PBS for collection).
Furthermore, I have observed significant decrease in number of cells in my more recent experiments. I have not been able to collect 100000 events during FACS anymore.
I know in part my lack of experience in the collection procedure is responsible for the inconsistent data, but I am starting to build confidence in my technique. I would like to know if there should be any changes or suggestions I can implement to hopefully produce a more noticeable difference between the Saline control and the Thioglycollate control.
Thank you for your help!