Neutrophils activation- HELP

[Mailendj]

HI ALL!!!
I'm new and I've found a lot of interesting information in this forum... but also following your advices... I've a lot of problems with neutrophils isolation from whole blood... so I need your help!!
I'll try to describe briefly the question:
For my PhD I have to analize bovine neutrophils production of ROS (by chemiluminescence) when they are treated with different stimula.
I have tryed to mantain all procedure at 37°C but neutrophils, without any stimulus, produces ROS, so I think that there is something that activate cells.
Following your advices, I try to to mantain all procedure at 4°C but the reult is the same... why???
Brief description of the procedure:
- Samples of blood are collected in tubes with EDTA as anticoagulant, and mantain cold in a thermic bag during transport,
-1:2 diluition with PBS
- Ficoll Paque (1077) density gradient (1500 g x 30 min)
- RBC lysis with NH4Cl-TRIS HCl buffer untill the solution become dark (about 5 min).
-2 washes with PBS (500g x 10 min)
-Pellet is in HBSS
-Total cell count/Tripan Blue

Guyssssssssss I need HELP....




Thank's

P.S.: Be patient with my English...


Milena

[luisillo]

I have some tips:
- Why don't use polymorphoprep to isolate neutrophils?
- You should wash with cold solutions to keep neutrophil adherence to tube walls to minimum.
- Neutrophils may produce basal amounts of ROS, you always need to have a negative control (without stimulus) in order to be able to measure the amount that is produced because of your stimulus

[Mailendj]

Thank's for the answer!!!
My question is.... all these change in temperature... blood at 37°C - PBS at 4°C-Density gradient phase at 20°C - assay at 37°C... don't stress neutrophils... it doensn't provoke loss of viability or spontanueous activation of cells????
Following protocol that I describe... negative controls produce a basal level of ROS, so I can't undestand if it is normal.... or it is caused by isolation!!!

[Warthaug]

I have isolated neutrophils for many years. Polymorphoprep is the w to go if you are preparing small amounts; its not so good for larger quantities of blood (>20ml, IMO). I'd add at this point that your method varies greatly from my own, so I've included a link to my own below. But generally speaking:

1) Do the whole isolation, including density centrifugation, at 4C. They only time you should warm is at the end, in preparation for your experiment.

2) Your activation is most likely occuring during your lysis step. 5min in that media will piss of the neutrophils royally.

My protocol:
[Only registered users see links. ]

Hope that helps,


Bryan

EDIT: I forgot to add, but chelators (EDTA/EGTA) are not the best choice for neutrophil preps. I precer citrate (ACD), but heparin works well too. Chelators deprive neutrophils of ions they generally need, thus stressing the cells, and as such, re-introduction of those ions at later time points can lead to activation.

[Mailendj]

HI....
...I'm here again with another question...
What is the better/most used composition of PBS (1X) for neutrophils???
In literature, papers don't describe it!!
I'm using:
PBS 10X
→ Dissolve the following in 800ml distilled H2O.
• 90g of NaCl
• 11.5 g of Na2HPO4
• 2,3 g of NaH2PO4
→ Adjust volume to 1L with additional distilled H2O.
→ Adjust pH to 7.2

Thank's for advices

[Warthaug]

PBS is 0.9% saline plus phosphate to buffer - ignore the recipes with KCl, etc, in them. Your recipe looks fine - except you should be pHing to 7.4. 7.2 is only needed if you filter sterilise, as the pH tends to rise ~0.2 units during filtration.

Alternatively, you can buy pre-made PBS. In some cases this is a better (but more expensive) option, but you get a more consistent product.

Bryan

[luisillo]

The recipe used in my lab is similar, but we add KCl, too. What is the deal with KCl? Does it interfere with any cellular mechanism? By the way, we have never used it with neutrophils, but with PBMC and sometimes with tumoral cell lines.

[Mailendj]

Another doubt: It's a problem if I adjust pH with HCl??? ... pH value of my PBS 1X after diluition fron 10X is 7.80... I think it's not a good idea to introduce new ions on my solution, as Cl-!!!!

[Warthaug]

HCl is the best way to go. Keep in mind you've already got a boatload of Cl in there from the NaCl. A little more will not affect things much. Use as concentrated a solution as you can, to minimize dilution of the PBS.

Bryan

[Mailendj]

Thank's Bryan for your precious advices!!!!