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Neutrophils - Science topic
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Questions related to Neutrophils
I’m new to the field of immunology and don’t have any prior experience working with neutrophils.
I’ve been isolating neutrophils from whole blood, lysing the cells directly after isolation with RIPA buffer (including protease and phosphatase inhibitors), and running the samples on a 10% SDS-PAGE gel. However, I’ve encountered some confusing results. Specifically, I’m not getting any signal for actin as a loading marker, which is even more puzzling than the inconsistent results I’m seeing with my target protein band.
I’ve tried using different actin antibodies as well as samples from different donors, but both times I didn’t observe a band for actin. Interestingly, other bands appear when probing with the target protein antibody, so I know there is lysate in the well.
Do you have any idea why this might be happening? I’d greatly appreciate your help.
I can't seem to find many protocols for isolating and culturing neutrophils specifically. Has anyone successfully done this or have any advice on how to do so?
I am facing challenges in isolating neutrophils from human peripheral blood after addition of Histopaque 1077. Neutrophils at the bottom of the tube after centrifugation are mixed with red blood cells. I am using hemolysis solution. The pellet containing red blood cells and granulocytes was resuspended in a hemolysis solution (150 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH 7.4) for 10 min at room temperature followed by centrifugation for 10 min at 4°C and 1600 rpm. At the end I have obtained the death of neutrophils and red blood cells. Could someone help me? Any suggestions?
Thanks all.
Some paper mentioned this receptor is expressed by monocytes and neutrophils but not NK cells. Then why Promega decides to use mouse FcgRIV for mouse ADCC assay?
Dear all,
I am planning to isolate neutrophils from whole blood of pigs for later RNAseq analysis. The samples may be taken over the course of several hours, so I assume I will have to stabilize RNA to get the transcriptomic state at the moment of sampling.
My plan so far is to add RNAlater to the blood immediately after sampling. Neutrophils will then be isolated via Lymphoprep gradient centrifugation and the neutrophils suspended in RNAlater prior to RNA extraction.
Potential concerns:
- when freezing whole blood stabilized with RNAlater, the sample tends to clot. Would this be a concern over the course of 1-2h at RT?
- does the Lymphoprep density gradient still work when RNAlater is added?
- any other concerns or alternative ideas?
Thanks a lot!
Has anyone performed the MTT test with human neutrophils? How long neutrophils should be incubated with MTT?
Hi there
I would appreciate some advice on RNA isolation from stored human neutrophils with the ultimate goal of RNA sequencing. Through various approaches it seems to be a trade-off between quality and quantity of the RNA I obtain. The neutrophils are stored at -80°C in RNAlater.
I prevent the initial clumping of the cells by processing with QIAzol lysis reagent. Downstream processing with Qiagen kits deliver an OK quantity of RNA, but for some reason the RIN values are always low (<4). I have switched to phenol/chloroform extraction (a published protocol) with isopropanol precipitation (have tried ethanol too) but this method does not seem robust because of the downstream ethanol wash steps and the risk of losing the often invisible pellet. With the latter approach I manage to improve RIN values but at the risk of low concentrations that do not help with downstream purposes.
I have tried the above with both healthy donor and actual clinical samples and get comparable results. I do not count cells (samples have been stored for a few years, and to minimise manipulation) but do extrapolate that the count should be sufficient based on matched PBMCs. I have to admit, I have tried many suggestions online and protocols, but to no avail. I was just wondering whether anyone else have had similar issues, and possible (last) avenues I could pursue.
Thanks in advance!
Hi all,
I've been working with HL-60 cells for ~2 years in our lab. I've been able to successfully differentiate them to a neutrophil-like phenotype using DMSO (1.3% final concentration, 5 days of treatment).
However, I'm having trouble finding how that method to differentiate them was discovered.
How was it discovered that DMSO differentiates these cells to a neutrophil-like phenotype?
Is the mechanism of DMSO differentiation understood?
Thanks!
Hi, scholars.
I am planning to conduct mouse neutrophil and T cell co-culture in Vitro and observe the effect of neutrophils on T cells.
I don't have any protocol. Could anyone provide me protocol and the reagents such as do I need to add any recombinant cytokines or peptide or anti-CD3 and CD28 to the culture medium. How long should I culture the T cells and neutrophils to T cell ratio.
Thank you.
How can I synthesis a lyse solution that I can lyse blood cells in 30 sec?
I made one type but the number of Granulocytes are shown in neutrophils in the plot.
I recently discovered that BFA is used in flow cytometry, enabling the observation of intracellular cytokines that would be otherwise released. Is the same principle applicable to ROS? I would like to see them in neutrophils.
Thank you
Hi, I am using 2 gradients Percoll protocol to isolate neutrophils from whole blood. I prepare gradients of desired density myself on the day of the experiment from Percoll (Sigma 1644). In my recent trials after centrifugation the blood stays on top of both of the gradients (normally it should be separeted into parts and placed in between the gradients). I assumed the Percoll concentration must have been higher than I had calculated. I've replenished my solution and tried again several times and still got the same result. I think my commercial Percoll Stock solution (which is kept at +4C) might have gone off. Is there any way to verify if the source of my problem is faulty Percoll?
Dear all,
Hello, I am recently working on isolating neutrophils from murine bone marrow by using Histopaque 1099 and histopaque 1077, but the purity of the cells are always unsatisfactory. Here I have several questions concerning the techniques. Firstly, which layer is neutrophils, could anyone help to illustrate this with photos or figures. Secondly, I am wondering what other techniques could be used to help to improve the overall quality of the cells.
Thank you so much for answering this questions for me. Best wishes to all !
I want to distinguish neutrophils from other cells in PFA-fixed and paraffin-embedded tissues. Could you provide recommendations for a basic histology staining method, other than H&E staining, that would be suitable for this purpose?
I have isolated neutrophils in inactivated state as per experimental need. I am searching for neutrophil cryopreservation protocol. I have used FBS+5%DMSO for cryopreservation. But after revival of neutrophils (in RPMI 1640) from cryopreservation, I am hardly getting 40% cell survival. Most of them are activated due to stress, cell death and rupture of granules. I have also tried HBSS (without Ca2+ and Mg2+) still I am getting same result. What should I do in order to get inactivated neutrophil after cryopreservation?
I've tried to use Ripa buffer containing 0.1% SDS + 10% of proteinase inhibitors combined with 3 cycles of freeze/thaw (Dry ice with isopropanol/Dry bath 70ºC), followed by 16.000rcf for 10 minutes. My issue is, after all this process my samples look like "jelly". Has somebody experienced this issue before? How can I solve that?
I want to prepare NBT solution, but I don't know how much NBT powder to dissolve in PBS. Also, I don't have a BSA, can I do the NBT test without a BSA?
I am isolating RNA from human neutrophils with the goal of performing RNA-Seq to assess the transcriptome under various conditions.
I have been isolating neutrophils from human blood using the Miltenyi Maxpress whole blood neutrophil isolation kit. I lyse the neutrophils (apx 10 million cells) in RLT buffer with 1% 2-BME and I pipette vigorously to lyse the cells and them store in -80 overnight. The next day I extract RNA using the Quaigen RNeasy kit, and while the quality has (sometimes) been ok, my yield is too low (1000ng if I am lucky)
Does anyone have any recommendations for how to improve my yield?
I have tried the Trizol-Chloroform method as well, and the resulting RNA was unusable (260/280 of 1.4).
How much do neutrophils, microglia, and other immune cell activation and infiltration play a role after craniocerebral trauma?
I am trying to understand whether my isolated neutrophils are functional or not after isolation. Therefore, I would like to compare the functionality of freshly and cryopreserved neutrophils, especially in the context of their pro-tumorigenic properties. What methods would you recommend?
Thank you so much for your help.
Hi,
I want to use functional enrichment analysis to see which gene sets are enriched in my eluted samples. The eluted proteins were isolated from human neutrophilic cell lysate. When using the human proteome as background, the enriched terms are suspiciously neutrophil related so we want to use a subset that contains only the proteins detectable in human neutrophils to have a better comparision.
Do you know any site where i can find either a list of proteins or raw proteomic data of neutrophils from healthy adult humans?
I want to isolate mouse bone marrow neutrophils to study neutrophil extracelluar trap, but the vechicle group always activation.
The objective:
To compare the antimicrobial peptides compositions in neutrophils with stimulation and without stimulation.
Does anyone have any suggestions or protocols for visualizing isolated neutrophils from whole blood? Just a basic morphology stain perhaps?
A microscoping imaging of intestinal tissue was made, and macrophages and neutrophiles were detected.
I am trying to quantify automatically the number of neutrophiles (polylobed nucleus) on Qupath but i don't know which parameters to apply to quantify them.
Do you have any ideas please?
Trying to calculate how much blood I would need to draw in order to get a sufficient number of neutrophils (from which extracellular vesicles will be obtained).
I am isolating neutrophils using density gradient from human whole blood.
We are finding difficulties in neutrophils activating and clumping once resuspended either from a fresh sample or a frozen/thawed sample. Sample is acquired from human blood using Sepmate tube and gradient purification.
No calcium or magnesium is being introduced to the cells at any point during the purification process and current buffer being used is 0.5% BSA in PBS + 2 mM EDTA.
Sample is being thawed at 37C and washed in buffer at the sample temperature prior to being centrifuged at 300g for 5 minutes.
I'm grateful for any help, thank you!
Antibodies with alloimmune reactivity are likely to be present in the plasma of the multiparous women. It include antibodies to human neutrophil antigens (HNA) or to human leukocyte antigens (HLA) that could be detrimental to recipients who have the corresponding antigens, which is passively transferred during transfusion, as specified in the report of National Library of Medicine (2012). What is the likelihood of having blood transfusion related reactions from implicated donors, especially multiparous women?
Sachs, U. J., Link, E., Hofmann, C., Wasel, W., & Bein, G. (2008). Screening of multiparous women to avoid transfusion-related acute lung injury: a single centre experience. Transfusion medicine (Oxford, England), 18(6), 348–354. Retrieved from https://pubmed.ncbi.nlm.nih.gov/19140817/
Hi all,
Has anyone perform neutrophil depletion and adoptive transfer experiments?
To determine the function of one neutrophil receptor, I performed the following neutrophil adoptive transfer experiments. For the depletion of the neutrophils, CD45.1 mice were injected with 0.2 mg anti–Ly6G mAb (clone 1A8, BioXCell) by tail vein 24 hours before the adoptive transfer of neutrophils from CD45.2 mice. 24 h later, 1×10^7 bone-marrow derived neutrophils in PBS from CD45.2 mice were injected to CD45.1 mice via the tail vein. The proportion of peripheral CD45.2 neutrophils was measured by flow cytometry at the subsequent 24 h and 48 h, and surprisingly, almost no CD45.2 neutrophils were present.
I wondered whether it was because the residual antibodies were depleting CD45.2 neutrophils or because neutrophils had a short life span.
Thank you very much!
I have a TEM image of a mouse bone marrow neutrophil. Can someone explain what is in this image?

In an article regarding IHC usen in myeloid sarcoma there was a report on a positive reaction to neutrophil esterase (Granulocytic sarcoma of the lips: report of an unusual case Badri Srinivasan. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:e34-e36), but i'm not sure if it is he same (synonym) to chloracetate esterase.
Thanks.
I want to study effect of particular protein of PMN on parasite load in mice. In this set of experiment I am planning to adoptively transfer PMNs from wild type mice into parasite infected knockout mice. Then I want to compare parasite load between non adoptively transfer vs adoptively transfer vs control wild type mice. Please help me in executing answer to this problem.
Hi every one, I have questions about neutrophil adherence.
I isolated neutrophils using Polymorphprep.
After isolation, I seeded it to 12-well plates with RPMI 1640 medium (+10%FBS, 1%PBS) and stimulated it with LPS.
Methodologically, it is supposed to attach to the culture plate after activation.
However, I found that both unactivated and activated neutrophils not strictly attach to the plate at all, it's almost all suspension state.
When I check the inflammatory cytokine with the qPCR, it seems that the neutrophils were activated successfully.
But I wonder is it not normal for this unadherent neutrophils?
Do anyone have experiences with this, please share with me.
Thank you so much.
I did IHC prior using markers MPO(neutrophils), F4/80 (macrophages), and CD3(T-cells) in mouse laryngeal tissues. However, I hardly saw any staining. Are there alternative markers for these cells that anyone would suggest?
I should stimulate a neutrophils culture to undergo apoptosis as a positive control for Annessin V kit.
Which is the best way to induce apoptosis in a short time?
Thanks
Does anyone have experience checking autophagy flux with LC3B antibody (western bot) in neutrophils from mice bone marrow?
Dear colleagues,
I need to isolate Neutrophils from human blood using percoll.
If you have experiences with this, could you please sharing your protocol?
Besides, neutrophils of course could survive short time after collection, but I need to keep it for a while in medium and treat it with LPS to check some gene expression.
Could I ask about the appropriate medium for Neutrophils and how long it could survive then?
Thank you.
I isolated the bone marrow neutrophil by B6. Cells received tBHP stimulation and detected the intracellular MPO expression by flow Cytometry. Although neutrophils decreased the expression of MPO in the intracellular after tBHP stimulation, neutrophils without any treatment produce low level of MPO. The B6 mice was born in November 2021.I'm not sure whether age is related to neutrophils function in B6. Who had the experience. Please share with me.
Sincerely
Hi!!
I have done some deep research for specific monocyte and neutrophil markers for qPCR (receptors, etc) but the literature is incredible biased.
Right now I am using the following markers:
Monocyte specific: TLR7, CCR5 and CX3CR1
Common markers for neutrophils and monocytes: CCR2, TLR2, CCR1
Neutrophil specific: CD177 and Neutrophil elastase
Would you feel this is adequate or is there any marker that I am really missing?? Because my data is a bit confusing and I am thinking that maybe there are better markers for these immune cells specifically.
Thank you!!!
I want to co-culture Neutrophils with leukemia cells (non-adherent) and want to separate the Neutrophils from the leukemia cells after the co-culture. If Neutrophils are purely adherent then I will be able to do that.
In the NETosis the primary granules present inside neutrophils are ruptured causing the release of NE and MPO that assist producing NETs inside neutrophil nucleus. My question is how excatly this azeurophilic granules are releasing these proteases? some paper says it is directly fusing with neutrophil nucleus and some say both NE and MPO has nuclear localization signal.
I want to know by what exact molecular mechanism is causing this release. Is there any surface receptor present on primary granules? or if it is directly fusing with neutrophil DNA which proteins and receptors are involved?
Hi guys, I hope you all are doing well
I just wanted to know wheter i can publish an experimental protocol design based only in previous literature (I have not performed it experimentally). If so, could you recommend some journals which I could submit my paper? It is a protocol for studing the intracellular and extracellular responce of human neutrophils against a yeast.
Thank you in advance
I am part of a lab doing flow cytometry on blood samples from mice inflicted with TBI. Does anyone have a protocol for this? Also, any suggestions on markers/antigens to screen for?
Dear all,
I am trying to isolate PBMCs from human blood. I am doing a gradient centribugation to remove most of the erythrocytes. The leftover erythrocytes I get rid of them using ACK lysis buffer for 3 minutes at RT. All steps I do at RT and very gently. I am using HBSS Ca-/Mg- with BSA 0.5% and Human FcR block from BD as blocking solution, for 20minutes at room temperature. I do the antibody incubation at RT for 30minutes in the dark. When I analyze by FACS the P1 population I get a double granulocyte population that stains positive for classical neutrophils markers (see picture attached). I assume the second population are activated neutrophils. Have anyone have experience this too? Any idea to get more consistent PBMC isolations? Thanks. Miguel
I am trying to fixate some neutrophils to study extracelullar traps but I'm having issues where the cells contrast is very low or no cell is present on the slides on the scanning microscope. I am fixating the cells with 4% PFA then 4% Glutaraldehyde, 1% Tannic acid, finalising with alcohol dehydration. Most of the protocols I see use Osmium Tetroxide, but I am avoiding OsO4 because of its toxicity. My question is if my problem with absent cells or poor constrast is due to not using OsO4 or there might me another issue? Does anyone know a protocol for fixating such sensible cell as neutrophils that does not uses OsO4?
Hi everyone,
I have been trying to induce NET in mouse neutrophil for a while with no success. I wonder if anyone can help me trouble-shoot? This is what I do.
I isolate neutrophil from bone marrow using protocol described by Swamydas et al. (Curr Protoc Immunol. 110: 3.20.1-3.20.15), which involves (1) Flush marrow cells into RPMI (no FBS) (2) RBC lysis with 0.2% NaCl for 20 seconds (3) Separation with Histopaque 1077 and 1119 (4) Resuspend in RPMI (no FBS).
I then, in an Eppendorf, add (1) PMN (2) PMA 100nM (3) Sytox 200nM, then aliqote 200uL into each well, incubate in 37C, 5% CO2 for 3 hours before I read. Each group is in triplicates.
I have 3 groups, one untreated, one PMA-treated and one Triton-treated. The Triton-treated has very high Sytox signals, showing Sytox works. But no difference is found between resting and PMA-treated.
Once I use DNAase and brought down the signals in the untreated, so I thought maybe they are already activated. But when I look under confocal, there isn't much NET formation either.
Has anyone has similar problems in the past? Thanks so much!
I am trying to isolate neutrophils from mouse bone marrow with percoll (62%). The goal is to stimualte the isolated neutrophils with LPS and afterwards lyse the cells for RNA analysis. I wonder what is the best temperature to work with during isolation (on ice or room temperature) since I read that neutrophils are not easily responding when put on ice? However working on room temperature comes with a cost of activation during isolation?
I also wonder who is experienced in isolating RNA from these cells?
I'm doing experiments on neutrophils producing NETs. In the preliminary experiment, I successfully induced NETs from rat neutrophils using PMA. When I used ADP preactivated platelets to stimulate neutrophils, NETs were produced much less than in the PMA group. And the cells would cluster together after the stimulation, which I think might affect the fluorescence intensity.
I wondered whether platelets and neutrophils needed to come from the same rat, and if so, whether platelets in the resting blood would be altered during neutrophils isolation?
By the way, Neutrophils are taken from bone marrow and I used Sytox Green to indicate the formation of NETs.
Your advice and experience are greatly appreciated!
I have used Stem Cell kit (EasySep Direct Human Pan-Granulocyte Isolation kit Cat# 19659) to purify PMN/neutrophile from fresh human whole blood (anticoagulant is EDTA, ), the isolated PMN stocked in Falcon 5 ml polypropylene round-bottom tube, about 4 ml cell suspension per tube , store in 4◦C for about 2 hours during the purification work, then spin down at 1200 rpm, 25◦C for 4 min, discard supernatant and resuspend the pellets, pooling all suspension together, I continued to count cells which take about 5 – 7 minutes, from last week and this week, I found the pooled suspension medium become very viscous and glue-like medium during the counting, and I lost more than 90 % of purified PMN, I would like you help me to troubleshot the issue here.
Dear Colleagues,
I want to culture neutrophils isolated by human blood samples. Can someone please provide insights into which method is the best for culturing neutrophils?
Looking forward
Thank you in advance
I am trying to isolate RNA from freshly isolated neutrophils from adult blood. Further I maintain culture for 3 hours hours according to my need. I use trizol choloroform method. I got very poor yield, A260/280 is also less than 1.8 even. No result was obtained on agarose gel. Can anyone please share your protocol or suggest me a good idea what will i do. Thanks in advance
Recently, I purify murine bone marrow neutrophil through 3-layer Percoll Gradients recently(72%-62%-56%), the purity is so low, between 40%-60%. I always follow the following steps:1. Grind the tibias and femurs in HBSS, filter the cell suspension with 40um strainer, wash 3 times; 2. Layer the cell suspension on 56% Percoll, centrifuge at 1500g for 30min with no break at RT; 3.Discard the frist two layers and harvest the third layer between 62% and 72% into RPMI; 4. Wash with RPMI 3 times. 5. Double stain CD11b and Ly6G to detect the purity.
I did not lysed red blood cells, is this step necessary? The cell layer is clear, as the picture showed, and I am very careful when harvest the neutrophils, but the number of neutrophil I got is always 1.5 to 2 times than other protocol mentioned, at such a low purity. Can anyone give me suggestion to got higher purity?


Can activating neutrophils with IL-8 at different concentrations show different responses?
hello
I am studying neutrophil and need specific marker for activated one ( nucleic or cytoplasmic markers) for FACS, IF applications. Thank you
I was wondering if anyone has any experience using alternatives for FCS in their HL60 cell culture (or similar cells), I've read that they can sometimes be cultured serum free. However long term I was wondering if there were any alternatives.
I'm happy to do a side by side comparison myself, but any starting input would be great.
Thanks for any help
What is the best way to draw plot ( Graph) to present engraftment data
Which is normally include the time it takes for platelets to reach >20 and neutrophil to reach >0.5 ?
I want to present my data in a conference yet? I am not sure what is the best plot?
As we know, long-term overnutrition, including consumption of a high-fat, high-fructose diet (HFD), can induce chronic low-grade inflammation in mice. However, we find a signifant low count of leukocytes as compared to the normal level. In detail, both lymphocyte and monocyte are significantly more reduced by HFD out of normal range, but not granulocyte. Generally, inflammation can increase leukocytes, especially neutrophils; however, our experiment showed different results. What are the conceivable reasons and mechanisms?
Neutrophile isolation kits or density gradient method
Hej, I want to activate neutrophils and collect their released granular content. I would like to do it without any biological (LPS etc.) or chemical (PMA etc.) stimuli.
I tried harsh pipetting and vortexing of the cells, with a following incubation of 15min, then spin to collect protein content. However, the yield was fairly low.
Is there a more efficient way? Maybe longer incubation time? I would like to avoid total lysis of the cells, but maybe I triggered that already with my treatment?
Any suggestions are highly appreciated!!!
Hi everyone,
I have found a few articles that give recipes for making cell culture media specifically for human neutrophils. I would like to do experiments where I would procure and magnetically isolate human neutrophils and then observe their behavior the same day or one day later at the latest. Given this, please share your thoughts. Here are a few other q's: Is RPMI or IMDM a better base media? Does it even make a difference given the short timeframe? Is 10% FBS or 20% FBS better? Is L-glut necessary?
Thanks so much for your input!
Human and animal studies have found increased serum Uric Acid levels are caused by Fluoride.
Uric Acid and its salts are known as a danger signal for activation of the NLRP3 gene (Nucleotide-Binding Oligomerization Domain, Leucine Rich Repeat And Pyrin Domain Containing 3) coding for the Inflammasone immune response leading to CASP1-catalyzed IL1B and IL18 maturation and secretion.
Uric acid also increases IL-1β, IL-2, IL-6, and TNF-α.
Monosodium Urate crystals induce Neutrophils to form Neutrophil Extracellular Traps (NETs), associated with severe COVID-19.
Uric Acid level could help to explain risk factors for severe COVID-19, such as Kidney disease and Diabetes.
African Americans are more at risk for Gout and Covid-19.
In the initial description acute leukemia was not seen in patients with VEXAS. An elderly male with GCA has presented with megaloblastosis and cytopenias raised inflammatory markers and neutrophilic dermatosis. Can it be seen with VEXAS?
Any suggestions on how to remove neutrophils in the frozen thawed cord blood samples? I'd like to enrich for progenitors and monocytes in the blood sample. I have used Miltenyi columns and StemCell EasySep for the enrichment but would like to see if there is any way to get rid of neutrophils so the progenitors and monocytes are enriched in the sample. Thank you.
Hi! I have been processing whole blood for several years now, using Ficoll and Dextran sedimentation. However, I am about to receive new samples from pregnant women that have cesarean delivery at night and so blood would be stored for a few hours until I can get it and process it.
Do you think this is a viable option? Or should I just stick to the patients that have cesarean delivery during the day? In case I DO decide to use them, should I store the whole blood at room temp or in the fridge?
Thanks for your answers in advance. I appreciate any comment on this subject.
I need to analyze and quantify inflammatory cells in different mouse tissues, people have indicated me to use ImageJ but I have not been able to use it so far, could someone help me with the steps
We are working on human neutrophils and we need to test the degranulation pathway using p-Hck. Any suggestion for an antibody that works with western blot?
Does anyone know if there are any distinct markers for N1 and N2 neutrophils that work with flow cytometry?
I am working with cultured mouse neutrophils, purified using negative selection (StemCell kit). They seem to have high viability (<10% death after 20h in culture), compared to what someone else has shown in another lab (60% death after 20h in culture). Does anyone have experience with this, and knowing what would be an expected viability?
Hello,
I am studying neutrophil-platelet aggregates in whole blood samples for a project. Can I use MPO for neutrophils (we have also CD11b, CD15 and CD16) for neutrophil-platelet aggregates? (Since for MPO I should use intracellular staining, I was wondering if it harms platelets?)