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What is the right thioglycolate to use to harvest mouse macrophages?

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mlfraga
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Topic Started by mlfraga
on 6/17/2009 8:25 AM   
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Can anybody provide the exact reference to purchase thioglycollate? (or thioglycolate... Are they different???)  I've been searching in different pages and forums and I can't determine which product is the right one to use. Sigma sells a bunch of different kinds and I've read some post where people report 3% sodium thioglicolate killing the mice...

Thanks


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samm
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Posted By samm
on 6/17/2009 11:59 AM   
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Hi! Just use the search feature on scientistsolutions.com for a very informative discussion thread on this subject. 3 or 4% thioglycollate is most commonly used, and you can just get it from Sigma. Try to store the solution for as long as you can before use.

All the best,

-sam

 

Quoting from the previous thread,

Aged 4% thioglycollate in saline works very well. Ready made formulations tend to differ, but the simple recipe with Sigma thioglycollate has worked just fine. The 'elicited' macrophages differ from 'native' ones, but they do not have an 'activated' macrophage phenotype.
The aging part can be done by storing the autoclaved sterile thioglycollate in the refrigerator over several weeks (2-3 atleast), preferably several months. I'm not absolutely sure why the aged version is better, but theres years of anecdotal data from labs all over the world on this one!

 



mlfraga
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Posted By mlfraga
on 6/18/2009 2:27 AM   
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Hi,

thanks for the quick responses.

 

Samm, you mention:

samm said:
Aged 4% thioglycollate in saline works very well. Ready made formulations tend to differ, but the simple recipe with Sigma thioglycollate has worked just fine. The 'elicited' macrophages differ from 'native' ones, but they do not have an 'activated' macrophage phenotype. The aging part can be done by storing the autoclaved sterile thioglycollate in the refrigerator over several weeks (2-3 atleast), preferably several months. I'm not absolutely sure why the aged version is better, but theres years of anecdotal data from labs all over the world on this one!

 

Could you be more specific about the "simple recipe with SIgma thioglycolate" please? Sigma has 16  hits under thioglycolate so I still don't know which is the one I should use. I keep finding contradictory information in the literature as to whether I should use sodium thioglycollate, broths, mediums, etc...

 

So far, what I gathered from your various posts is that you prepare thioglycolate by diluting in water a 2x solution. To prepare you recommend 2 cycles of autoclave (10 min 105ºC) with o/n 4ºC storage in between. "Aging" should be at least for 2 weeks in the fridge. At the time of use, i.p. injection of 1x solution (4%) in saline (1 ml max volume). Is all tihs correct?

 

To collect naive macrophages, should I just do a peritoneal lavage with cold 30% sucrose solution?

 

Thanks for your help

 

Marta


Last edited Jun 18, 2009, 4:45 AM by mlfraga

mlfraga
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Posted By mlfraga
on 6/18/2009 2:44 AM   
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By the way, do you also have a reference provider for one of these vendors that sell the ready made aged thioglycolate?

 

Thanks

 

Marta



R Bishop
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Posted By R Bishop
on 6/19/2009 7:56 AM   
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Marta,

I havent ever seen "pre-aged" thioglycollate.  I spoke with an Immuno lab down the hall and here is there protocol and response.

"I actually got the Thioglycollate from Fisher, Difco makes it, but Fisher distributes it.  It is not real easy to find on their website, but the catalog number is below along with the recipe for making the broth.  

 
I autoclave 500ml, aliquot it out, and store it in the dark.  I was told that the older it was, the better it worked.  Not sure if that is really true, but that is what people believe.  It will still work just fine if you make it new.  Also, be sure to keep it sterile if you are going to store it for a long period.  
 
 
 
Fisher Cat # DF0430-17-1 
 
*Thioglycollate Broth:
10.1 g Brewers thioglycollate (DIFCO)
add 250 ml distilled water
Boil for 1 minute to dissolve
autoclave to sterilize
 
Inject 1 ml intraperitoneally
1-3 days later, most of the peritoneal exudate will be neutrophils and 4-6 days after injection, 98% will be macrophages, which can be isolated out of the peritoneal cavity with ice-cold HBSS."
 
 
She say you will still get a response even if it isnt aged all that long.
 
Hope that helps
 
Rus
 

"Everything should be made as simple as possible, but no simpler." -- Einstein



samm
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Posted By samm
on 6/19/2009 16:02 PM   
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Hi Marta! Take a look at Rusty's post - I've never personally used the Difco thioglycollate, but have heard good things about it. An alternative if you wish to use a broth based activation are Sigma cat #70157, which works pretty well - the formulation is slightly different from Brewer's TG broth, but both contain the same amount of TG. As Rusty mentions, this works best if its aged after autoclaving, but *works* immediately too.

If you want a more defined prep, use Sigma T0632 - autoclave a 8% stock in water and add 2x sterile saline (some folks do it all together - 4% in saline, but thats sometime tricky). Store for atleast a fortnight at 4dC for optimal results - older the better.

To answer the other Q you had - 'naive' macrophages can be obtained by peritoneal lavage - 30% sucrose (ice cold) worked best for me. 'Elicted' macrophages - obtained when a Thioglycollate injection i.p. is done 72-96 h earlier are greatly enriched populations caused by selective migration - they do not have an activated/angry phenotype, but are not entirely naive. You'll typically get 8-12 fold more macs/mouse after elicitation.

Finally, if you do the same process for 4-12 h, you'll get a greatly enriched neutrophil population - some of which will have NETs (look really pretty!)

All the best

-sam

 



mlfraga
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Posted By mlfraga
on 6/22/2009 0:45 AM   
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Thanks guys!!!

 

All your comments were really helpful.

 

Cheers :-)

 

Marta



TheGrad
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Posted By TheGrad
on 3/4/2010 11:27 AM   
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Samm,
I have been trying to use Sigma's Brewer Thioglycollate. I aged it for a couple of weeks, then tried injecting 2ml into 6-8wks old mice. When I collect the peritoneal lavage 4 days later, I am only able to recover about 10million cells total, and if I do a FACS analysis directly ex-vivo I am consistently getting about 50% cell death when staining with 7AAD, and only about 25% of the cells being MAC1/F480 double positive!!!

Have you ever seen/heard of that happening? I have tried in about 15 mice already, and they're all giving me the same result.
My thioglycollate is in PBS, and I am flushing the peritoneal cavity with cold PBS+2%FBS before staining for FACS.

Any feedback would be appreciated, I am pretty confused at this point!!!

Thank you!!!

ps: My TG is starting to turn green....should I be worried?



shadi
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Posted By shadi
on 5/18/2010 10:57 AM   
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Hello Samm

I have just started using thioglycollate (Sigma T9032) to elicit peritoneal mouse macs.  If i do not use the thioglycollate cells look uniform but if I induce with thio, I have  a lot of clumps of cells that I can not break and the pellet looks granular when resuspended.  

I have used anywhere from 1-3ml 3% thioglycolate for 2-3 days and over and over i have the same problem.  I have also used two different strains of mice with the same result.

I need to count these cells and with clumps it is very inaccurate. To me it looks like there are epithelial cells with macs attached to them.

Does anyone know? I would greatly appreciate any ideas.


Last edited May 18, 2010, 16:16 PM by heehawmcduff

samm
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Posted By samm
on 5/18/2010 13:59 PM   
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How are you doing the lavage? Is the pH/osmolarity of your thioglycollate okay? See previous posts for preparation instructions.
Ideally,
~3 d post instillation of aged TG,
CO2/nembutal anesthesize the mouse,
carefully make a Y-incision leaving peritoneal membrane intact,
instill in ~4-5 ml of ice-cold 30% sucrose in peritoneum using a 26/27 g needle.
The peritoneum swells up like a balloon - gently palpate for a few seconds.
Aspirate out using a 20 gauge needle - make an incision in the peritoneal membrane to get the remnants of the lavage.



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