Hello, what is the duration of your LPS treatment? 

I used 14-18 h for LPS induction of ROS, in macrophages. 

I am not sure if neutrophils responds to LPS rapidly by producing ROS. 

The standard induction stimulants for neutrophils are PMA (as you know) and zymosan, which is TLR2 ligand, whereas enterobacterial LPS is TLR4 ligand. 

In addition, LPS dosage also matters in my experience. It should be between 100 ng/mL to 1ug/mL. 

Adaptive response or lack of effects have been reported, for low doses such as 10 to 20 ng/mL LPS. 

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There are papers that used some dose of PMA to prime neutrophils overnight, and then challenge neutrophils with LPS acutely (within 30 min), followed by an oxidative burst measurement. It is possible you try to find some of such papers and follow their methods. 

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I am not clear what you meant by "donor variability" and "donor dependent". 

LPS and PMA are inducers, not donors. An ROS donor, e.g., would be something like exogenous H2O2, or xanthine/xanthine oxidase. 

Best

Hi Nai-Kei,

Thanks for the response!

Perhaps I was not clear; my idea of donor variability refers to the difference in response by neutrophils isolated from different donors. I observed consistent neutrophil ROS response to LPS from some donors, while others exhibited no response up to a concentration of 100 ug/ml.

To answer your question, neutrophils respond rapidly to stimuli, usually within the 1st hour. Even without stimulation, ex vivo neutrophils will not be suitable for experiments after a few hours.

My protocol calls for continuous measurement every 3 min for 3 hours. 25 nM PMA results in rapid ROS production (within 15 min), and responders to LPS (10 ng/ml) start producing ROS after the 30 min mark. AUC is then derived from the curve for statistical analysis.

It will be great if there is a sure-fire way for neutrophils to respond to LPS after priming by other compounds (TNF-a perhaps?). Have yet to try out priming with specific cytokines and would appreciate any inputs from the community =D.

We recently used primary human neutrophils in our ROS (HOCl) imaging study. The neutrophils were isolated from a donor, and prepared using a protocol as described here: http://www.ncbi.nlm.nih.gov/pubmed/18226596

According to that protocol, after isolation and before use, the cells are kept in cold PBS (4C). 

Just before my experiment, I warmed the cells to 37C for 1h. Subsequently when challenged with PMA (200 ng/mL), the cells rapidly responded in like within 15 min. (PMA effects waned subsequently). 

Although the imaging quality was very good, for our publishing purpose, I have concerns about the 4C storage step, in PBS. 

Perhaps the low temp. can keep the cells in an metabolically inactive state (but there's no evidence from the paper). 

Otherwise, it is likely that leaving cells in PBS (for like a few hours) is sufficient to induce autophagic signaling. 

Starved cells are known to produce not so robust response in oxidative burst. (This has been reported, but not well studied or explained). 

GM-CSF may be important for priming neutrophils in this regard. 

See: http://www.ncbi.nlm.nih.gov/pubmed/12176910

TNF-a may be too general, and when its dose is too high, it becomes a death signal. 

Thanks Nai-Kei,

I usually re-suspend my cells in RPMI with 10% FBS and keep them on ice throughout. Neutrophils incubated at 37oC vs on ice has marked differences in activation markers, which is the reason why I choose to keep them on ice usually before use. Also, the cells are usually used within 2~3 hours after isolation.

Just came across a few papers on NADPH oxidase assembly and LPS/TNF-a priming effects. Will be trying out TNF-a as soon as it arrives and update here again =D.

100 ng/ml LPS is the upper Limit of LPS in blood of patients with severe Sepsis. Try to analyze blood neutrophils without purification (blood ROS Generation Assay), just in diluted citrated blood. Then zymosan at about 1-2 µg/ml Triggers blood ROS Generation and added LPS at about 1-10 ng/ml acts as a co-Stimulator.

Thanks Thomas,

Just a quick query - how is blood ROS generation assessed? As I need to ensure that it is the neutrophils at play, I am concerned that other cell types (i.e. monocytes, platelets) would interfere. Would you consider intracellular assessment of ROS using DCFH-DA and assessed by flow cytometry to be an option?

Neutrophils generate much more H2O2-singlet Oxygen that are detected by luminol reaction in a photon-multiplying microtiter plate luminometer . The few photons emitted by other cell types can be neglected.

"Would you consider intracellular assessment of ROS using DCFH-DA and assessed by flow cytometry to be an option?"

DCF is not selective for any specific ROS. It does not detect superoxide, in particular. 

See: http://www.jbc.org/content/278/5/3170.full

However, its use in FACS is generally acceptable. Your can try DCF after sorting, if that's what's being planned. Another way is first enriching your neutrophils using a standard protocol for isolation. Then compare that with whole blood response. 

Use of DCF in imaging is not advisable, despite reported attempts.

http://www.jbc.org/content/275/18/13175.abstract 

Thank you both for sharing! I have tried zymosan and it works perfectly, only I wish to continue using LPS as a control as it is more relevant in my study. I have come across some protocols where enriched neutrophils are first 37oC-primed before treatment and ROS measurement. I tried it once (15 min 37oC) and the cells can now respond to LPS and generate ROS (albeit slightly but significantly). Will certainly try whole blood in the near future and update this space =D.

Chun can you tell me how you prepare your LPS? I am using it as well but I am not getting any response from my retinal cells and I wonder if I am making it up incorrectly. I generally measure out the powder into a tube and dilute with DMEM to 1mg/ml stock. I vortex this for 5mins and then dilute it down as required.

Dear Chun, zymosan directly triggers ROS generation of neutrophils, LPS is only a costimulator.

Hi Reinold, sure. I got my LPS from Sigma from which I reconstitute directly from the lyophilized powder to 5mg/ml stock with sterile water. We dilute them down to the working concentration in the respective medium (for me I use RPMI + 10% FBS). Of note though, LPS may be adsorbed onto eppendorf tube surface, so we usually vortex for at least 15 - 20 seconds, though usually a stock of high concentration should not be much of an issue. Hope it helps :)

Hi Thomas, thanks for the inputs! From the last post, I have pre-warmed my neutrophils for at least 30min to 37degC before LPS challenge. It works more often than not now so it's good to note :)

I agree with Dr. Stief, the typical stimulant for neutrophils are zymosan (or whole yeast), fMLP (formyl bacterial peptide), or PMA (phorbol ester).  

However, if you find that LPS alone triggers some degree of ROS, that's interesting to know. Good luck. 

Have you ever measured ROS production with LPS stimulation after TNF-a priming?

Hi Ye Gao, thank you for the question.

I have not tried priming the neutrophils with TNF-a, though it crossed my mind once. However, due to the nature of our experiments, it was preferred that we do not introduce additional stimulants to the reactions. Till this day though, I still observed donor variability in their cells' ability to generate ROS in response to LPS, but at least they are responding. As mentioned, I heat-primed the neutrophils at 37oC for 30 min first.

The blood anticoagulant you choose is very important. Heparin itself is a neutrophil Stimulator, Citrate or EDTA might be better.

I am trying to stimulate neutrophils with PMA and in the future LPS but am starting with just PMA to work out the protocol. I treated my neutrophils with a range from 20 nM to 100 nM PMA for 4 hours at several cell concentrations. I see no effect at any PMA concentration, the elastase concentration is the same as untreated cells. I was thankfully able to see a difference in elastase activity that correlated VERY NICELY with cell number. So at least I know the assay is working. I just do not know why the PMA in the Abcam kit (ab235979) is not stimulating NET formation?

I was not sure if it could be the media - I used RPMI 1640 Medium, GlutaMAX Supplement, HEPE (Thermo Fisher). I read a paper saying glucose is important for NETs formation, but RPMI should have glucose so not sure what the problem is. Any suggestions would be helpful. I have also heard serum inhibits NET formation, but you can get heat-inactivated which would be ok to use. I used the BSA that was included with the Abcam kit because when I asked previouly, they confirmed it was heat inactivated. So, this should not be the problem although I could try not using the serum.  Any help would be much appreciated.

Purchase your LPS from Sigma (5 mg vial) and stabilize it with 5% human albumin of drug qualitiy.