I had prepared 4% thioglycollate (BD Biosciences, cat# 211716) in ddH2O, autoclaved and aliquoted in 15 ml sterile tube, and stored it at 4 dc in the dark around 8-9 months ago. During the time of preparation it was light brown to straw in color but now it has completely turned green, maybe due to oxidation. My questions concerning this are: (1) can I use this oxidized 4% thioglycollate to elicit murine peritoneal macrophage and is it toxic for macrophages, (2) will it affect the yield/quality of the macrophage, (3) why do people prefer to use aged thioglycollate over freshly prepared, and (4) can I use freshly prepared thioglycollate to elicit murine peritoneal macrophage?
1) yes, not toxic
2) it won't affect it
3) http://www.ncbi.nlm.nih.gov/pubmed/9050940
4) no (see 3)
1) yes, not toxic
2) it won't affect it
3) http://www.ncbi.nlm.nih.gov/pubmed/9050940
4) no (see 3)
This may seem trivial but how green is "green"? Like the colour of the researchgate theme green?
You can use fresh thioglycollate- but aged works better-- with fresh you will still get an abundance of macrophages.
Thank you all for your quick response and valuable suggestions
Tapas
Thomas Ashhurst .... I like your funny question.
It was the best example I could think of. The aged thioglycollate that I used was brown or straw coloured, not actually green. Perhaps the BD mixture has a different composition and so oxidisation does make it green, but I would be cautious, as thioglycollate supports bacterial growth very well. Swirl around you bottle/tube and check if you can see particles floating in a cloudy mixture.
If you wanted to be extra sure, you could add some to culture medium (RPMI or DMEM) and keep for 1-2 days at 37*C, and see if anything grows.
Hi Thomas Ashhurst
I already check thioglycollate broth in Complete RPMI didn't get any growth
Thanks
You should get a good harvest (5-7 million cells/mouse) of resident peritoneal macrophages without using thioglycollate.
Many labs use aged thioglycollate...as long as it is transparent green it is fine..but if it is slightly hazy then its time to through it away...you can use freshly brewed thioglycollate also..it can yield around 12-15 million macrophages after 72 hrs from C57BL/6 mouse.
Aged thio works the best. Did a time course experiment when we made a fresh batch (determined average yield for 5 mice) and found best yield after aged 5 months. Cells did not show increase in MHC-II, showed good expression of MAC-1, -2, and -3.
The green color will go away if you autoclave the TG again or heat it up briefly in a microwave (keep an eye on it to avoid messy spills).
Always be sure that your Thio is endotoxin free. Most lots are not and you will get variable maturation/activation dependent on endotoxin (LPS) contamination.
- Badges
- Science method