Problems inducing thioglycollate peritonitis

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AuCh
AuCh's picture
Problems inducing thioglycollate peritonitis

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!

Best,
Austin

heehawmcduff
heehawmcduff's picture
Hi there,

Hi there,

From looking at your method, I would have a couple of suggestions but these are anecdotal and from personal experience i.e. I haven't compared my methods to yours.

1. I would use autoclaved and aged thioglycollate.  You can use the brand new mixture but the aged solution seems to work better (or so I've heard).  If you age it for at least a week in the dark at 4oC that should be fine.  Also, you could try 4% thioglycollate rather than 3%.

2. I would wait a little longer to collect your macrophages, perhaps 48-72h.  This will skew your population to a more pro-inflammatory phenotype as you will have a greater proportion of 'inflammatory' macrophages that have migrated to the peritoneal region rather than the native population, but you will increase your yield. 

If you take a look at this thread on Scientist Solutions discussing peritoneal lavage, you can see that an ice cold 30% sucrose solution was used.  I have never tried this (I've always used ice cold PBS) but it may be worth a try to improve cell viability/reduce clumping. 

If you continually have problems with red cell contamination then Dextran sedimentation may help although optimising the technique is best.

I wish you better success with your retrievals.  Hope the advice helps.

BioCris
BioCris's picture
Hello Austin

Hello Austin
I also work with peritoneal macrophages, I hope to be able to help.

1. Autoclaved your thioglycollate.
2. Wait at least 3 days before extracting the cells.
3. Do not make cervical dislocation, sacrifices the mouse by lethal dose of sevorane, or with CO2 (if you do not have sevorane). This will help you with the blood infiltrated.
4. Use cold saline or cold PBS, inject 5 to 8 ml per mouse, and masage the abdomen, cuts the skin of the abdomen, so that you can see the peritoneum, removes the PBS using a needle (do not cut the peritoneum), this also will help to prevent the blood infiltrated.
5. Keep your samples in cold and processes it or culture the cells before it one hour of extraction.

My best wishes to you.