Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

neutrophil activation (MPO assay?)


  • Please log in to reply
6 replies to this topic

#1 Crumbled Ham

Crumbled Ham

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 13 January 2010 - 06:15 AM

I have been looking around for a good neutrophil activation assay. I know you can use myeloperoxidase (MPO) activity as a neutrophil activation marker but I wonder how I can measure this without having to buy expensive kits or antibodies.

Any suggestions? Or any ideas of other good assays I could use?

CH

#2 victorius

victorius

    member

  • Active Members
  • Pip
  • 23 posts
1
Neutral

Posted 10 August 2010 - 07:12 PM

You can assay peroxidase activity with a cocktail of Amplex red 100 uM (Invitrogen) and 10 uM H2O2 in phosphate buffer 50 mM, pH 6.0. Buy MPO from Athens Research to set up a standard curve in a microplate.

For isolated neutrophils peroxidase = myeloperoxidase, since there is no other peroxidase (like eosinophil peroxidase).
IN this case I guess you can also use any peroxidase substrate, like TMB...
The most popular I think is the colorimetric o-dianisidine assay, which I have not tried.

More specifically, you can measure chlorination activity of MPO with DTNB, although you have to prepare it. Or, Cayman Chem has a kit with a fluorescent dye that it says is selective for chlorinated species. Look up the kit, it costs 215$ but you can buy individual components too. You can always add sodium azide (1 mM ) to make sure you are measuring MPO and not something else. Or you can omit Cl- in your mix.

Vic.

#3 newborn

newborn

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 72 posts
2
Neutral

Posted 10 August 2010 - 07:45 PM

There is a cheap method to measure MPO

http://www.ncbi.nlm....pubmed/16316776

#4 shytownmed

shytownmed

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 30 August 2010 - 10:42 AM

You can assay peroxidase activity with a cocktail of Amplex red 100 uM (Invitrogen) and 10 uM H2O2 in phosphate buffer 50 mM, pH 6.0. Buy MPO from Athens Research to set up a standard curve in a microplate.

For isolated neutrophils peroxidase = myeloperoxidase, since there is no other peroxidase (like eosinophil peroxidase).
IN this case I guess you can also use any peroxidase substrate, like TMB...
The most popular I think is the colorimetric o-dianisidine assay, which I have not tried.

More specifically, you can measure chlorination activity of MPO with DTNB, although you have to prepare it. Or, Cayman Chem has a kit with a fluorescent dye that it says is selective for chlorinated species. Look up the kit, it costs 215$ but you can buy individual components too. You can always add sodium azide (1 mM ) to make sure you are measuring MPO and not something else. Or you can omit Cl- in your mix.

Vic.



Hi,

I am working on the same project of measuring sequestering on Neutrophiles using MPO Assay. I am a newbie at this method. I have tenatively developed a protocol using o-dianisidine as a subtrate. I have a question: I have a lysis buffer consisting of potassium phosphate, Hexa trimethylammonium Bromide (HTAB), and EDTA. The buffer seems to form a white precipitate after mixing the reagents. I am guessing that the source of the white precipitate is EDTA. Is this normal? Do I resuspend the precipitate before I add to tissue samples?

I also have substrate buffer consisting of potassium phosphate, hydrogen peroxide, and 0-diadinasidine diHCl. This buffer also forms a white precipitate. Is this normal as well? I think that the source of the precipitation is the o-diandinasidine.

Both buffer are at pH 6.

#5 victorius

victorius

    member

  • Active Members
  • Pip
  • 23 posts
1
Neutral

Posted 02 September 2010 - 09:53 AM

Hi, here are some suggestions: Try adding EDTA from a stock solution (eg 1M) rather than powder, then titrate to pH 6.0; EDTA needs a pH ~8.0 to get dissolved properly.
Also, HTAB does not get easily dissolved so leave the PB+HTAB mixing for a while in a shaker at RT. White flakes will form again after cooling, but mix again at RT and they will vanish. I have no experience with o-dianisidine.

Hope it helps,

V.

#6 shytownmed

shytownmed

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 06 September 2010 - 01:57 PM

Thank you so much for these great suggestions. I did make 1M EDTA from powder.

Shytown

Hi, here are some suggestions: Try adding EDTA from a stock solution (eg 1M) rather than powder, then titrate to pH 6.0; EDTA needs a pH ~8.0 to get dissolved properly.
Also, HTAB does not get easily dissolved so leave the PB+HTAB mixing for a while in a shaker at RT. White flakes will form again after cooling, but mix again at RT and they will vanish. I have no experience with o-dianisidine.

Hope it helps,

V.



#7 victorius

victorius

    member

  • Active Members
  • Pip
  • 23 posts
1
Neutral

Posted 13 May 2011 - 06:53 AM

I found that when assaying tissue sample homogenates with Amplex Red (for peroxidation) or APF (for chlorination) there is a "dilution effect" of sorts by which higher dilution of my samples yield higher fluorescence. It could be interference by 3 mM NEM (I use it to block GSH) or some stuff in the samples, I did not test further, but it's convenient to check several dilutions when using these dyes. If anyone has noticed this and has an answer, please post!

Thanks,

V.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.