Science topic

Neutrophils - Science topic

Explore the latest questions and answers in Neutrophils, and find Neutrophils experts.
Questions related to Neutrophils
  • asked a question related to Neutrophils
Question
4 answers
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.
Relevant answer
Answer
I had 52 million cells and BCA showed that I had around 2 ug/uL protein and I loaded 60ug per well. I think there was protein degradation as neutrophils expel a lot of proteases when activated. I have to find a way to avoid excess protein degradation.
  • asked a question related to Neutrophils
Question
2 answers
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?
Relevant answer
Answer
As noted above, isolation can be accomplished. A Percoll gradient should be considered following digestion, and my experience is with type 4 collagenase and DNase to digest organs. Proteases have the potential to strip membrane antigens, confusing subsequent flow cytometry analysis. Culture is a different challenge. PMNs are terminally differentiated and don't proliferate. Further, they have a short lifespan in vitro, from hours to a few days. The placenta has been shown to have myeloid progenitors, i.e., PMN precursors, which can be cultured. Any standard culture with growth factors for hematopoietic/myelopoietic progenitors would work. I would assume it would include GM or G-CSF, and any other cytokines could be included. SCF, FLt3L, IL3 etc. Question for another discussion.
  • asked a question related to Neutrophils
Question
3 answers
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.
Relevant answer
Answer
You are welcome, Rosemari!
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
I had the same question today. After diving into the literature, I have the following findings.
As you mentioned, mouse FcgRIV is reportedly expressed on monocytes/macrophages and neutrophils, but not NK cells (PMID: 16039578; PMID: 26497511). Checking FcgRIV expression in ImmGen confirms these findings. Therefore, because NK cells are the classical mediators of ADCC, it seems weird to use FcgRIV activation as the proxy for ADCC activity.
The best explanation I can find for Promega's decision is that other cell types, including macrophages, have ADCC activity (PMID: 34204268 and references therein), which could be mediated by FcgRIV. An upside to using FcgRIV is that it has high affinity for IgG2a/c and IgG2b, but no binding to IgG1.
The big picture is that none of these "bioassays" from Promega or other vendors is really a suitable substitute for in vitro (or in vivo) experiments with actual, primary effector immune cells. IMO, these assays are best understood as generic FcR activation assays, rather than indications of specific Ab-driven effector activity (e.g., ADCC, ADCP, etc.).
  • asked a question related to Neutrophils
Question
1 answer
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!
Relevant answer
Answer
Hello Bjorn Klabunde,
RNAlater is not recommended for preserving RNA in whole blood, plasma, or sera. Because of their high protein content, these fluids will form an insoluble precip-
itate if they are mixed with RNAlater.
Instead, you may use special tubes that contain a proprietary reagent for stabilization of intracellular RNA immediately upon collection. Intracellular RNA is stabilized for up to three days at room temperature. These tubes may be used for collection, storage, and transportation of blood and stabilization of intracellular RNA with subsequent isolation and purification of intracellular RNA from whole blood for further analysis.
You may want to refer to the links below for more information on these specialised tubes.
I am also attaching a few references that may help.
Best.
  • asked a question related to Neutrophils
Question
2 answers
Has anyone performed the MTT test with human neutrophils? How long neutrophils should be incubated with MTT?
Relevant answer
Answer
Dear Dr. Astrid Parenti
I have performed MTT assay but not with human neutrophils. You may incubate neutrophils with MTT for 2-4 hours. You may have to increase the incubation time with MTT until the purple color is evident inside cells when viewed under microscope.
There are investigators who have performed MTT assay with neutrophils. You may want to refer to the articles attached below. They will be helpful.
Regards,
Malcolm Nobre
  • asked a question related to Neutrophils
Question
1 answer
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!
Relevant answer
Answer
To get the highest yields and best quality RNA in your situation:
Add the appropriate volume of Trizol to your sample, and ensure the cells are completely lysed. Then, freeze the Trizol lysate overnight at -80C (this helps with lysis). Trizol gives slightly better RNA yields than QIAzol. Do not vortex at any point in the protocol, since this will shear long RNAs.
The next day (or later), isolate RNA from the Trizol following the manufacturer's instructions but with the following modifications:
1) after removing the aqueous phase, add it to 600 uL of chloroform, spin, and remove the aqueous phase again (ie do a chloroform extraction). This helps to remove guanidium salts. Using Phaselock tubes can help during this step, since the chloroform extraction won't have the interphase that you see in the Trizol extraction.
2) after adding isopropanol to the aqueous phase, add 1uL of GlycoBlue (or glycogen, if your downstream application is affected by a dye) and precipitate the RNA at 4C for 1 hour with rotation of the tube. Then, pellet the RNA with a 30min spin at 4C at 18k g.
3) do three washes of the RNA pellet with cold 75% ethanol instead of one.
4) resuspend the RNA pellet with as little as 5uL of NF water, or whatever volume gives you an acceptable concentration.
5) keep the sample on ice as much as possible throughout the protocol. The final RNA product should always be on ice or in the freezer (-80C for long term storage, -20C for short term storage).
Isopropanol precipitation will give you much better yields than using the Qiagen columns, and higher quality too. Your comment that it is not robust enough is not true, since every RNA lab in the world regularly performs isopropanol precipitations. Handling a small pellet takes a bit of practice, but with practice (and using glycoblue/glycogen) you can easily deal with pellets from <10,000 cells. You can practice with cultured cells or any non-important cells you have access to.
If you get more neutrophils in the future, I would recommend storing them at -80C as Trizol lysates. This is better for long term storage than RNAlater.
  • asked a question related to Neutrophils
Question
6 answers
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!
Relevant answer
Answer
Hi Drake,
I have the same questions, and i also could not find a fully reviewed paper about it, have you figure it out yet?
I tried to ask ChatGPT, and here is the answer, hope is useful for both of us:
Dimethyl sulfoxide (DMSO) is a solvent commonly used in cell biology experiments, and it can also induce the differentiation of certain cell types. For example, in the human acute myeloid leukemia cell line HL60, DMSO can induce these cells to differentiate into neutrophil-like cells.
The mechanisms by which DMSO induces differentiation primarily include the following:
  1. Activation of transcription factors: DMSO can alter the expression of certain genes associated with cell differentiation. It can activate or inhibit specific transcription factors, guiding cells into the differentiation pathway of neutrophils.
  2. Cell cycle regulation: DMSO may promote differentiation by affecting cell cycle regulation. It alters the progression of the cell cycle, making cells more likely to exit the proliferation cycle and enter a differentiated state.
  3. Regulation of signaling pathways: DMSO can influence intracellular signaling pathways. By changing the activity of these pathways, DMSO might promote the activation of key molecular pathways associated with neutrophil differentiation.
  4. Regulation of oxidative stress: DMSO can also modify the redox environment of the cell, which is an important regulatory factor in the differentiation process. Adjusting the oxidative stress state within the cell may help initiate the differentiation program.
Through these mechanisms, DMSO induces morphological and functional changes in HL60 cells, leading them to exhibit characteristics of neutrophils. These characteristics include morphological changes such as cell shape and nuclear maturation, as well as the upregulation of specific neutrophil markers.
  • asked a question related to Neutrophils
Question
2 answers
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.
Relevant answer
Thank you so much for your recommendations.
After isolating neutrophils, I would like to know know how to set up neutrophil and T cells co-culture methods. Do we need to add any recombinant cytokines, chemokines or Anti-CD3 and CD28 to the Neutrophil- T cell co-culture and how long should we stimulate and other parameters.
  • asked a question related to Neutrophils
Question
3 answers
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.
Relevant answer
One common approach involves using a hypotonic solution combined with detergents to disrupt cell membranes rapidly. Here's a simple recipe for a lysis solution:
Ingredients:
1. Distilled water (hypotonic)
2. Detergent (such as Triton X-100 or NP-40)
3. Salt (optional, for maintaining osmolarity)
4. Buffering agent (optional, for pH stability)
  • asked a question related to Neutrophils
Question
1 answer
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
Relevant answer
Answer
BFA is a reversible inhibitor of protein transport. Following treatment with BFA, the Golgi complex disassembles and redistributes into the endoplasmic reticulum within minutes. BFA is a potent, rapid, and reversible inhibitor of secretion. It inhibits the GTPase exchange factor acting on the ARF protein. ARF activates ADP-ribosylation factors to the Golgi complex.
BFA is mostly used in studies of membrane trafficking. It increases intracellular cytokine staining signals and is commonly used for intracellular staining of cytokines for flow cytometry. It blocks transport processes during cell activation and causes an accumulation of cytokines at the Golgi complex/ endoplasmic reticulum.
Besides its use in intracellular staining of cytokines, BFA shows antibiotic actions and induces apoptosis and autophagy in mammalian cells. The use of BFA to detect ROS is not known.
Nevertheless, if you are interested in measuring ROS production, the most widely used method is the use of the fluorescent probe 2’,7’ dichlorodihydrofluorescein diacetate (DCFH2-DA). This molecule is colorless and lipophilic. Diffusion of DCFH2-DA across the cell membrane allows it to be acted upon by intracellular esterases, which deacetylates it into DCFH2, rendering it cell impermeable. The actions of multiple types of ROS (hydrogen peroxide, peroxynitrite, hydroxyl radicals, nitric oxide, and peroxy radicals) on DCFH2 oxidize it into DCF which is fluorescent (reported Ex/Em: 485–500 nm/515–530 nm) and can be detected using a flow cytometer.
However, superoxide does not strongly react with DCFH2 but can react with another probe dihydroethidium (DHE) to yield the fluorescent product 2-hydroxyethidium as well as other fluorescent superoxide-independent oxidation products. The fluorescent products of DHE oxidation can be detected using an excitation wavelength of 518 nm and an emission wavelength of 605 nm. Include controls in the assay to have valid experimental results and conclusions.
You may want to refer to the articles attached below for detection of intracellular ROS by flow cytometry.
Best.
  • asked a question related to Neutrophils
Question
3 answers
How to count neutrophil
Relevant answer
Answer
Most cytometers use the impedance method for cell count.
Any change in the electrolyte state and distribution of electric charges in the cell suspension medium may interfere with the count.
Such could be the case after separation in density gradient. I recommend the washing of saline cells and their resuspension in compatible plasma or better in a mixture plasma-red blood cells (separated by centrifugation).
Let me know.
Alternatively and without additional manipulation the "old" Burker method could be, although time-consuming, resolutive
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
Before you come to any conclusion regarding the Percoll solution, you should try the protocol given below, if it is not similar.
1. Add 10mL of 75% Percoll into a 50mL conical tube using sterile pipette.
2. Carefully overlay 10mL of 62% Percoll on top of 75% Percoll using sterile pipette.
3. Dilute 10mL of fresh blood 1:1 in 10mL of serum-free RPMI 1640 without antibiotics.
4. Carefully overlay 20mL of diluted blood on top of the 62% Percoll using a sterile pipette in a dropwise manner.
5. Centrifuge the 50mL conical tube at 200 × g for 25 min and 400 × g for 15 min.
6. Using sterile micropipettes first collect PBMCs on top of the 62% Percoll layer.
7. Then collect neutrophils positioned between the 75% and 62% Percoll layers and transfer the cells to a new 50 mL conical tube.
8. Add RPMI 1640 to the tube up to a volume of 50mL. Wash the neutrophils by centrifuging at 300 × g for 5 min.
9. Remove the supernatant completely and resuspend the cell pellet in 1mL of neutrophil buffer.
10. Add 10μL of cell suspension to the hemocytometer and count the cell number under the microscope.
To prepare 62% Percoll gradient solution (50ml): Add 31ml (62%) of Percoll to 5ml (10%) of (10X) PBS and 14ml (28%) of ultra-pure water.
To prepare 75% Percoll gradient solution (50ml): Add 37.5ml (75%) of Percoll to 5ml (10%) of (10X) PBS and 7.5ml (15%) of ultra-pure water.
Some important points to be noted:
1. Percoll should be kept at room temperature (22°C–25°C), otherwise Percoll density changes and the gradient centrifugation success will be compromised.
2. The volume of diluted blood may be adjusted depending on the amount of blood drawn per donor. The amount of diluted blood overlaid should not be greater than 1.5 times the volume of the Percoll gradient. For instance, 15mL of drawn blood (i.e. 30mL of RPMI 1640 diluted blood) can be overlaid on a gradient of 10mL of 75% Percoll and 10mL of 62% Percoll.
Best.
  • asked a question related to Neutrophils
Question
3 answers
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 !
Relevant answer
Answer
If you may want to use Histopaque for isolating neutrophils, add 3 ml of Histopaque 1119 in a 15-ml conical centrifuge tube. Overlay with 3 ml of Histopaque 1077. Do not disturb the interface between Histopaque 1119 and Histopaque 1077. Overlay the bone marrow cell suspension on top of the Histopaque 1077 layer. Avoid disturbing the interface between the cell suspension and Histopaque 1077. Centrifuge for 30 minutes at 872 × g at room temperature without brake. Collect the neutrophils at the interface of the Histopaque 1119 and Histopaque 1077 layers.
For more information, you may want to refer to the article attached below.
If you may want to use Percoll for isolating neutrophils, the cells may be treated on a three-layer Percoll gradient of 78%, 69%, and 52% Percoll respectively, diluted in HBSS, and centrifuged (1500 g, 30 min, room temperature) without braking. The neutrophils from the 69%/78% interface and the upper part of the 78% layer may be harvested into 1% BSA-coated tubes.
For more information and diagrammatic representation, you may want to refer to the articles attached below.
Best wishes!
  • asked a question related to Neutrophils
Question
4 answers
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?
Relevant answer
Answer
Can be easily identified in H and E stained sections because if their multilobed nucleus and granular cytoplasm morphology. iHC can be done using CD10, CD15 and other markers for better differentiation.
  • asked a question related to Neutrophils
Question
4 answers
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?
Relevant answer
Answer
you can try the stem cell CS10
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
Try to follow bead beater or ultrasound as the first option. These presents high yield..Freeze thaw cycyles may conclude with low protein recovery. Anyway jelly visual must be dependent to genomic material composition of the cells...DNA and RNA molecules must be digested or removed during lysis. To perform this I suggest using DNase, RNase (Benzonase nuclease as cocktail)...
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
Hello Maryam Rahnama,
You may follow the below protocol.
1. Take 100ul of blood and mix with 100ul of 0.2% nitro-blue tetrazolium in saline and 100ul of PBS. You may incubate this mixture at 37°C for 15 min and then leave it at room temperature for 15 min.
2. You may make smear on a glass slide and fix with methanol for 3 min and then stain with Pappenheim's stain for 3–5 min. Neutrophils that ingest the dye and have an oxidative burst create large black formazan deposits in the cytoplasm.
3. You may run this assay in conjunction with blood from healthy control subjects to identify any problems that you may face with specimen handling.
Best.
  • asked a question related to Neutrophils
Question
3 answers
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).
Relevant answer
Answer
I agree with Matthew, I used TRIzol RNeasy hybrid for neutrophil and it gave me good RNA quality, but i would use more neutrophil if you could
  • asked a question related to Neutrophils
Question
3 answers
How much do neutrophils, microglia, and other immune cell activation and infiltration play a role after craniocerebral trauma?
Relevant answer
Answer
Franz Schelling, thank you for your valuable guidance. We will be more rigorous in studying the immune-inflammatory response after craniocerebral trauma.
  • asked a question related to Neutrophils
Question
6 answers
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.
Relevant answer
Answer
You might find the paper An easy and reliable whole blood freezing method for flowcytometry immuno-phenotyping and functional analyses of interest. The authors report that following freezing 20% of the neutrophils are lost on thawing (presumably lysed) and of the remaining neutrophils 42% were dead/dying. "FSC low PMs were CD15 + CD16+ live/dead dim dying neutrophils (mean frequency: 42% of total PMs) (Figure 2(b)).
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
Where can I find list of human organs that work together?
  • asked a question related to Neutrophils
Question
1 answer
I want to isolate mouse bone marrow neutrophils to study neutrophil extracelluar trap, but the vechicle group always activation.
Relevant answer
Answer
Dear Dr. Chen Shuai
In the below attached paper, the investigators isolated neutrophils from murine bone marrow by negative immunomagnetic isolation using a complex antibody cocktail. Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro.
You should try this antibody cocktail which is able to isolate highly pure mature neutrophils which remain in a resting state during the isolation protocol.
Regards,
Malcolm Nobre
  • asked a question related to Neutrophils
Question
2 answers
The objective:
To compare the antimicrobial peptides compositions in neutrophils with stimulation and without stimulation.
Relevant answer
Answer
Polymorphonuclear leukocytes (PMNs or neutrophils) extraction from blood:
  1. Collect a blood sample from a healthy individual using a sterile syringe and needle. The volume of the blood sample will depend on the specific experiment and the number of neutrophils required.
  2. Transfer the blood sample to a sterile centrifuge tube containing an anticoagulant, such as ethylenediaminetetraacetic acid (EDTA) or heparin, to prevent clotting.
  3. Gently mix the blood and anticoagulant by inverting the tube several times.
  4. Centrifuge the blood sample at a low speed (e.g., 200-300 x g) for 10 minutes to separate the blood components. This step will result in the formation of three layers: a top layer of plasma, a middle layer of white blood cells (including neutrophils), and a bottom layer of red blood cells.
  5. Carefully collect the middle layer of white blood cells using a pipette and transfer them to a new sterile centrifuge tube.
  6. Add a red blood cell lysis buffer (e.g., ammonium chloride-potassium (ACK) lysing buffer) to the tube containing the white blood cells to remove the red blood cells. Incubate the tube for 5-10 minutes at room temperature.
  7. Centrifuge the tube at a low speed (e.g., 200-300 x g) for 10 minutes to pellet the white blood cells.
  8. Discard the supernatant and resuspend the white blood cell pellet in a suitable buffer, such as phosphate-buffered saline (PBS) or Hank's balanced salt solution (HBSS).
  9. Count the number of neutrophils using a hemocytometer or an automated cell counter. Adjust the cell concentration as needed for your experiment.
Degranulation of PMNs:
  1. Prepare a stimulation buffer by adding a suitable concentration of a stimulant, such as phorbol 12-myristate 13-acetate (PMA) or N-formylmethionyl-leucyl-phenylalanine (fMLP), to a buffer solution (e.g., PBS or HBSS).
  2. Transfer the desired number of neutrophils (from the previous step) to separate sterile tubes. Label one tube as the "stimulated" sample and the other as the "unstimulated" sample.
  3. Add the stimulation buffer to the "stimulated" sample, while adding an equal volume of the buffer solution (without the stimulant) to the "unstimulated" sample. Incubate both samples for a specific time period (e.g., 30 minutes) at 37°C to allow degranulation to occur in the stimulated sample.
  4. After the incubation period, centrifuge both samples at a low speed (e.g., 200-300 x g) for 10 minutes to pellet the neutrophils.
  5. Collect the supernatant from each tube, which will contain the released antimicrobial peptides from the degranulated neutrophils.
  6. Store the supernatant samples at -80°C or proceed with the analysis of the antimicrobial peptides using suitable techniques, such as radial diffusion assays or mass spectrometry, to compare the compositions of the peptides in the stimulated and unstimulated samples
  • asked a question related to Neutrophils
Question
2 answers
Does anyone have any suggestions or protocols for visualizing isolated neutrophils from whole blood? Just a basic morphology stain perhaps?
Relevant answer
Answer
You may check the neutrophils by May Grünwald-Giemsa staining based on their characteristic morphological appearance (for instance, there is a single nucleus, which is multi-lobed, and can have between 2 and 5 lobes).
The neutrophil pellet suspended in 100μL PBS is cytocentrifuged at 300 × g for 3 min and then fixed. The cells are spun onto the slide, stained by May Grünwald-Giemsa staining, and examined by light microscopy.
You may prepare the required reagents as follows.
Preparation of May Grunwald stain
  1. Dissolve 0.3 g of May Grunwald dye in 100 mL absolute methanol in a 250 mL conical flask.
  2. Warm the mixture to 50°C in a water bath for a few hours and allow it to cool to room temperature.
  3. Stir the mixture on a magnetic stirrer and leave it stirring for 24 hours.
  4. Filter the mixture and the stain is ready for use.
Preparation of Giemsa stain
  1. Add 1.0 g of Giemsa dye into 66 mL of glycerol and warm the mixture in a conical flask for 1-2 hours at 50°C.
  2. Cool the mixture to room temperature and add 66 mL of absolute methanol.
  3. Leave the mixture to dissolve for 2-3 days, mixing it at intervals.
  4. The stain is ready for use after you filter.
Staining protocol:
  1. Prepare an equal volume of May Grunwald stain and phosphate buffer pH 6.8. Mix well and pour onto the slides to fully flood the slides. Stain for 10 minutes.
  2. Prepare a 1:10 dilution of Giemsa stain with phosphate buffer pH 6.8. Mix well.
  3. After 10 minutes of May Grunwald staining, pour away the May Grunwald stain off the slides.
  4. Then pour the Giemsa mixture onto the slides and stain for another 15 minutes.
  5. After 15 minutes, pour off stain and flush the slides with running tap water.
  6. Clean excess stain with kim wipes.
  7. Air dry the slides. You may use the hair dryer to dry the slides but use it at the lowest speed and no longer than 10 seconds each time.
  8. Place the long cover slip on the area of interest.
  9. The slide is now ready for examination by microscopy.
You may also want to refer to the article attached below. It may be helpful.
Best.
  • asked a question related to Neutrophils
Question
3 answers
Method to identify NETosis
Relevant answer
Answer
I'm not sure if you are just looking for a method to isolate neutrophils or methods to assess NETosis; I think the above answer gave a lot of options to measure NETosis. The link I'm providing here is a good way to isolate human neutrophils:
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
You should stain the sections with CD15 first which facilitates the identification of PMN in the histological section. In my experience with Qupath, you need to count the PMN manually and the problem is which intestinal tissue are we talking about and if from an experimental model or humans (i.e. small bowel or large bowel biopsies, sections of a Swiss roll from the entire colon). This aspect is important for comparison of the sections of the study. Another aspect is the position of the neutrophils (mucosal surface, membrane, serosa).
I hope it helps to improve the accuracy of the determination.
  • asked a question related to Neutrophils
Question
2 answers
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).
Relevant answer
Answer
In the past we've had consistently good yields using isopyknic focusing on a continuous gradient of colloidal poly(vinylpyrrolidone)-coated silica (Percoll).
Described in: Methods in Enzymology vol. 133 pages 459-460 (1986)
(1) (PDF) Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis (researchgate.net)
  • asked a question related to Neutrophils
Question
3 answers
I am isolating neutrophils using density gradient from human whole blood.
Relevant answer
Answer
If I understand correctly, this is the sample you want to use for your proteomic analysis. Therfore, you don't want to bring in media or serum, as this would totally mess up your proteomics!
Wash the cells 3x with PBS and transfer them into the Eppendorf tube for freezing of the cell pellet.
Later, you will use your lydis buffer and prepare the sample for the analysis.
  • asked a question related to Neutrophils
Question
1 answer
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!
Relevant answer
Answer
Hi Charles, My name is Eilysh and I am a Product Manager at STEMCELL Technologies. Thank you for your question! Please refer to our guide on how to thaw frozen primary cells: https://www.stemcell.com/technical-resources/how-to-thaw-frozen-primary-cells.html. Additionally, we do sell frozen neutrophils. They can be successfully frozen and thawed and then used for downstream assay but their lifespan is limited (less than 6 hours). If you want to use frozen neutrophils, it needs to be immediately after thawing and for something short. Ideally, we would recommend sticking to freshly isolated neutrophils if possible.  I hope this helps! If you have any more questions, feel free to email us at techsupport@stemcell.com. Kind regards, Eilysh
  • asked a question related to Neutrophils
Question
1 answer
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/
Relevant answer
Answer
In a study conducted by L. Thurn, et. al., entitled, "Incidence and risk factors of transfusion reactions in postpartum blood transfusions", the increase of HLA antibodies observed in most pregnancies, especially in multiparous women may increase the risk of transfusion reactions during pregnancy.
Another study, entitled, "Transfusion-related mortality: the ongoing risks of allogeneic blood transfusion and the available strategies for their prevention" by E. C. Vamvakas, et. al., suggests that in 65% to 90% of TRALI cases, WBC (including class I and II HLA or neutrophil-specific) antibodies have been identified in the plasma of the implicated donor and that most of these implicated donors have been multiparous women.
Thurn, L., Wikman, A., Westgren, M., & Lindqvist, P. G. (2019). Incidence and risk factors of transfusion reactions in postpartum blood transfusions. Blood advances, 3(15), 2298–2306. https://doi.org/10.1182/bloodadvances.2019000074
Eleftherios C. Vamvakas, Morris A. Blajchman; Transfusion-related mortality: the ongoing risks of allogeneic blood transfusion and the available strategies for their prevention. Blood 2009; 113 (15): 3406–3417. doi: https://doi.org/10.1182/blood-2008-10-167643
  • asked a question related to Neutrophils
Question
5 answers
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!
Relevant answer
Answer
Hi,
Maybe try to stain your BM-derived neutrophils with Zombie Dye Agua or PI to make sure whether they are alive when transferred to the recipient.
Maybe you could try parabiosis, instead of the adoptive transfer to achieve your goal. In that case, the recipient will receive donor-derived neutrophils all the time.
Regards!
Qian
  • asked a question related to Neutrophils
Question
3 answers
I have a TEM image of a mouse bone marrow neutrophil. Can someone explain what is in this image?
Relevant answer
Answer
The large darker structures on the right and along the bottom is the cell nucleus. The central vacuole is likely a lysosome that appears to contain the remnants of another cell that was phagocytosed. The remaining cytoplasmic details are not clear at this magnification and may be the result of sub-optimal fixation. The black spots are fixation/staining artefacts.
  • asked a question related to Neutrophils
Question
2 answers
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.
Relevant answer
Answer
Histologically, granulocytic sarcoma (GS) undergoes various morphological changes in the cells. It is composed of immature cells of the neutrophil granulocytic series. The infiltration of immature, poorly differentiated cells with round to oval nuclei is the predominant finding. Crystalline, rod-like, intracytoplasmic acidophilic bodies called Auer rods are sometimes seen. With these histological findings, it is hard to distinguish GS from other lymphomas including histiocytic lymphoma, lymphoblastic lymphoma, Ewing's sarcoma, lymphocytic leukemia, undifferentiated carcinomas, and primitive neuroectodermal tumors.
You may refer to the article attached below.
Anti-lysozyme and chloroacetate esterase can be used to diagnose GS.
There are two types of esterases, specific and non-specific. The specific esterase will enzymatically hydrolyze naphthol AS-D chloroacetate liberating a free naphthol compound. This then couples with a diazonium compound, forming highly colored deposits at sites of enzyme activity. This enzyme is usually considered specific for cells of granulocytic lineage. This specific esterase stain, chloroacetate esterase (also called Leder stain), stains neutrophils, neutrophil precursors, eosinophils, and mast cells and confirms the granulocytic nature of the tumor.
On the other hand, the nonspecific esterase activity (alpha-napthyl acetate esterase) is seen in monocytes. The cells of granulocytic series are negative for the nonspecific esterase activity.
So, in the article regarding IHC used in myeloid sarcoma in which there was a report on a positive reaction to neutrophil esterase means that the author is referring to the specific esterase namely, chloroacetate esterase.
Best.
  • asked a question related to Neutrophils
Question
1 answer
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. 
Relevant answer
Answer
Was your experiment going well? I am also conducting neutrophil adoptive transfer experiments, but I found that after injecting neutrophils from WT mice into KO mice that had depleted neutrophils, the circulating WT neutrophils in the KO mice quickly disappeared. I would like to ask if you have encountered similar problems and how to solve them?
  • asked a question related to Neutrophils
Question
2 answers
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.
Relevant answer
Answer
The neutrophils should attach using the protocol that you're using. However, if you really need them to attach, you can poly-l-lysine coat your plates. The neutrophils stick within a couple of hours.
  • asked a question related to Neutrophils
Question
4 answers
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?
Relevant answer
Answer
Thank you for all your suggestions Konstantin Benno Bräutigam , Malcolm Nobre Lukas Bolini . Trying to restain with some of these suggested markers.
  • asked a question related to Neutrophils
Question
4 answers
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
Relevant answer
Answer
There is no need to induce apoptosis in human cultures neutrophils, they go into apoptosis spontaneously after 24 hours. Do you wish to get a shorter time?
  • asked a question related to Neutrophils
Question
1 answer
Does anyone have experience checking autophagy flux with LC3B antibody (western bot) in neutrophils from mice bone marrow?
Relevant answer
Answer
May contain the relevant information.
  • asked a question related to Neutrophils
Question
3 answers
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.
Relevant answer
Answer
Geoffrey Garcia Thank you so much for your protocol.
Could you clarify a little bit more about your protocol:
1. Usually, how much is the amount of blood you will take for each sample? And after collect the samples, you could keep your samples in 4 degree fridge for how long before start isolation?
2. dilute Percoll 9mL of Percoll with 1mL of NaCl 1,5M to do a 100% of Percoll solution
--> Could I dilute Percoll in HBSS instead?
My lab is using Percoll PLUS (Cytiva)
2.Deposite your 1mL of WBC on the top slowly and centrifugate 3000rpm during 10min without break
--> some protocol I know mentions centrifugation in 20mins, do you think it is ok with longer time of centrifugation?
3.Could you tell me more about the wash step?
4. I need to keep neutrophils alive, maybe several hours at least after treating with LPS just to check some gene expression.
So do you think medium of HBSS+/+ (with Ca and Mg) + SVF is enough for the survival? Actually, I'm thinking of using cell culture medium as RPMI + FBS +P/S as normal cell culture protocol.
I look forward to your response.
  • asked a question related to Neutrophils
Question
1 answer
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
Relevant answer
Answer
The immune responses and associated gene expression is known to decline with age.
  • asked a question related to Neutrophils
Question
4 answers
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!!!
Relevant answer
Answer
There is considerable redundancy in the expression of chemokines, their receptors and TLRs. Thus, one might have concerns with the ones noted for monocytes. Speaking from a flow perspective; monocytes would be well recognized based on CD14, CD68 and CD115. Elastase should work for neutrophils. You will need to be specific as to which enzyme. CD15 would also be a consideration.
Having made these recommendations, myeloid cells have numerous subsets. For example MDSCs and dendritic cells. Also progenitor cells, such as found in the marrow or during inflammatory conditions can confuse issues. You might be best served with single cell analysis using multiple markers.
  • asked a question related to Neutrophils
Question
2 answers
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.
Relevant answer
Answer
Thank you for the detailed answer Malcolm.
  • asked a question related to Neutrophils
Question
1 answer
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?
  • asked a question related to Neutrophils
Question
3 answers
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
Relevant answer
Answer
It is difficult to publish a prposed protocol, framework or even a model in any journal without at least a pilot experiments. You should proof the applicability and efficiency of your concept with some results.
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
Dear Leroy,
In the paper cited below are some markers demonstrating the activation state of neutrophils. This is a study in human patients but you can easily adopt the protocol to mice. CD16 and CD18 are rapidly upregulated upon stimulation of the neutrophils. The systemic inflammation after TBI should be enough to induce the upregulation of those receptors.
It is Importen that work consequently unter cold conditions. The heparinized blond sample should be immediately coole down to 4 degree Celsius. Start the ytaining procedure within 20 minutes. Add 5 microliter antibody (for example cd16 fitc) to 50 microliter coole blood, mix carefully and incubate 20 min at 4 degrees in the dark. And 2 ml cold hypotonic erythrocyte lysing solution and incubate for Addition 10 min AT 4 degrees and in dark. Centrifuge 5 min AT 4 degree Celsius (1500 RPM with Standard Lab centrifuge, Rotor diameter 14 to 15 cm). Discard supernatant, wash once with cold PBS w/o Ca,Mg. Discard supefnatant and fix with 1 % Formaldehyde in PBS w/o. Wash once again with PBS w/o and resuspend the Pellet in 250 microliter PBS w/o and start measurement as soon as possible.
Best
Dirk
Holzer K, Konietzny P, Wilhelm K, Encke A, Henrich D. Phagocytosis by emigrated, intra-abdominal neutrophils is depressed during human secondary peritonitis. Eur Surg Res. 2002 Jul-Aug;34(4):275-84. doi: 10.1159/000063071. PMID: 12145553.
  • asked a question related to Neutrophils
Question
1 answer
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
Relevant answer
Answer
I could be missing something, but typically when you do a gradient centrifugation for PBMC prep, nearly all of the granulocytes and erythrocytes should be removed in the pellet. What kind of gradient are you using? I typically layer 20ml of washed (50% DPBS) whole blood on to 10ml of Ficoll-paque plus in a 50ml conical. ACK or any downstream removal of non-PBMC is not really necessary and our neutrophil contamination is <0.5% of purified PBMC.
  • asked a question related to Neutrophils
Question
7 answers
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?
Relevant answer
Answer
I had not worked with neutrophils, but for at least 95% of biological specimens Os is not needed for SEM specimen preparation. Neither cell attachment nor contrast are influenced by Os. Os was introduced to SEM protocols more than half century ago, when TEM protocols were adapted for SEM needs. So, Os migrated in SEM field, where it is not needed.
  • asked a question related to Neutrophils
Question
15 answers
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!
Relevant answer
Answer
Hello all, I found the discussion very helpful, and I am having trouble about stimulation of netosis as well. I was wondering if Luke C Davies any idea. I am using PMA/ionomycin cocktail for stimulation of NETs formation but I don't think it is working. I tried to incubate cells at 37°C in an incubator and stimulated those cells with 2 uL of PMA/ionomycin cocktail in 500 uL of RPMI medium. I couldn't see NETs formation, I saw in the literature people have been using PMA quite much and ionomycin as well. However, I didn't see any paper that use a cocktail like that at the same time and I was wondering if this is supposed to work, or there is sth else that I miss?
  • asked a question related to Neutrophils
Question
2 answers
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?
Relevant answer
Answer
Hi,
Isolation of neutrophils using percoll should be done at RT for sure. Once you isolate, put the cells in media with FBS if you want to stimulate them with LPS. Within 2-3 hrs, cells get adhered. After you are done with the stimulation, you can use this kit for isolation of RNA (considering these cells are sensitive and low life span):
PicoPure™ RNA Isolation Kit, Catalog number: KIT0204.
Good luck!
  • asked a question related to Neutrophils
Question
7 answers
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!
Relevant answer
Answer
Thank you for offering such valuable advice for me!
Best!
  • asked a question related to Neutrophils
Question
5 answers
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.
  • asked a question related to Neutrophils
Question
3 answers
I need to know how to measure micro RNA 223 in neutrophils
Relevant answer
Answer
You can run a RT-PCR experiment to measure the mir-233 Level . The primer sequence is given in an article which is ;
miR-223 forward, 5′-AGC CGT GTCAGTTTG TCA AAT-3′; reverse, 5′-GTGCAGGGTCCGAGG TC-3′
  • asked a question related to Neutrophils
Question
2 answers
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
Relevant answer
Answer
Neutrophils are extremely short-lived and sensitive cells in vitro. Here's a useful review on the topic with some good cell-line alternatives.
  • asked a question related to Neutrophils
Question
3 answers
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
Relevant answer
Answer
RNA extraction from sperm-challenged PMNs and control groups
The PMN cells exhibiting NETosis were collected and re-suspended in 100µl of PBS in 1.5 ml MCTs. The cells were mixed with 900 µl of Trizol reagent (Invitrogen) as per manufacturer’s instructions. The RNA pellet was dried in the air followed by dissolved in 30 µl of DEPC treated water and placed in a heating block at 55ºC for 5 min. Extracted RNA was quantified using a NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The quality and integrity of extracted RNA were assessed by running 200ng of RNA (heated at 65°C for 1 minute) in non-denaturing TAE buffered 1.2% agarose.
  • asked a question related to Neutrophils
Question
6 answers
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?   
Relevant answer
Answer
HI Christine Youn
Have you solve the problem in neutrophil isolation?I am also facing with the low purity using 81% and 63% Percoll gradients. I will be greatful if you can share your experience.
  • asked a question related to Neutrophils
Question
2 answers
Can activating neutrophils with IL-8 at different concentrations show different responses?
Relevant answer
Answer
Shin Murakami Thank you very much for your studies, professor! I used the references you sent me and I understand a lot.
📷
📷
  • asked a question related to Neutrophils
Question
12 answers
hello
I am studying neutrophil and need specific marker for activated one ( nucleic or cytoplasmic markers) for FACS, IF applications. Thank you
Relevant answer
Answer
Dear Iman Aldybiat,
You may look over the following info:
  • asked a question related to Neutrophils
Question
2 answers
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
Relevant answer
Answer
Fcs alternatives for HL 60 neutrophil like cell line is FPS (slightly different).
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
It would be better to use a line graph with markers of days on the abscissa and quantitative indicators of cell composition on the ordinate, on my opinion.
  • asked a question related to Neutrophils
Question
3 answers
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?
Relevant answer
Answer
i think the answer for that observation is pretty simple.
high fat diets increase the mass of adipose tissue. some studies have shown that increasing adipose tissue mass leads to decreased plasma leptin hormone concentrations. also, other studies have shown that leptin stimulates haemopoiesis.
so in summary; high fat diet increase adipose tissue mass, thus decreases leptin, decreases haemopoiesis, and eventually leucokyte and monocyte counts.
  • asked a question related to Neutrophils
Question
11 answers
Neutrophile isolation kits or density gradient method
Relevant answer
Answer
Could you tell me how to harvest the neutrophil layer with a Pasteur pipette in details? After centrifugation, I found the two leucocyte bands were too close and it was difficult to harvest the lower band of polymorphonuclear cells perfectly. So the purity of the neutrophils I got was very low.
  • asked a question related to Neutrophils
Question
1 answer
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!!!
Relevant answer
Answer
I have several suggestion:
You could trying thermal shock (45 to 1 to 45 celcius as fast as you can) or osmotic shock (distilled water for a minute). You can try to place your neutrophils in starvation for some times (i suggest 12 to 24h given the short half life of neutrophils, longer would lead to mainly apoptosis residues).
You could try physical membrane permeation, without lysis. maybe with a low intensity sonication or low voltage electroporation (pretty much as they do in molecular biology, but without inserting a plasmid or anything). It may works since it would allows a greater entry of calcium in the cell, which play roles in degranulation.
  • asked a question related to Neutrophils
Question
1 answer
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!
Relevant answer
Answer
You cannot maintain neutrophils for more than 24 hrs. because after 24 hrs. apoptotic cells are observed (more than 50%). RPMI 1640 will be a better basal medium with 10% FBS. No need to add L-glutamine.
Best.
  • asked a question related to Neutrophils
Question
12 answers
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.
Relevant answer
Answer
Geoff Pain an interesting correlation and sophisticated question. Look, what I would like to present is a simplified iron (fe) perspective while trying to link with all of your proposals and observations. First, fluoride appears to cause iron overload:
Uric acid is suggested as a telltale signal of early iron overload
Finally, instead of a causative role, uric acid appears to be the way the body attempts to mitigate excess iron since it is a powerful heavy metal chelator
"Urate acts as a chelator for iron and, in turn, iron can modulate the activity of xanthine oxidase and the production of urate"
As we are aware covid-19 actively tries to increase the availability of serum iron levels to replicate faster
It would seem that high initial uric acid levels would signal the increase of iron or the fact that the patient has some form of iron overload in the first place. Then at the next stage, Covid19 would attack the areas that produce uric acid thus removing its chelating effects. Uric acid is synthesized mainly in the liver, intestines and vascular endothelium. So it is no surprise that
COVID-19-associated gastrointestinal and liver injury - Nature
Covid-19 is a vascular disease:
All of these mean that Hypouricemia would be the end game.
"studies have shown that serum uric acid concentrations were markedly lower in patients with severe COVID-19 disease"
Now I need a little help with why attacking the kidneys would be advantageous
but nobody is perfect;-)
Lancet: COVID-19 Virus Can Attack Kidneys, Speeding Death
Sincerely
Prof Christopher G YUKNA
PS Fernando Kemta Lekpa African Americans suffer from gout more often and have higher serum iron levels than the rest of the American population which in the context of this discussion may have been due to adapting to deal with the malaria parasite. Just an idea but the research supports the hypothesis.
  • asked a question related to Neutrophils
Question
5 answers
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?
Relevant answer
Answer
No, but here may be a relevant paper:
  • asked a question related to Neutrophils
Question
4 answers
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.
Relevant answer
Answer
Thank you Willem for the suggestion. I think it is a good idea to deplete with CD15 beads. I will try it out. Thank you.
  • asked a question related to Neutrophils
Question
3 answers
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.
Relevant answer
  • asked a question related to Neutrophils
Question
6 answers
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
Relevant answer
Answer
As far as I know, you can use an ImageJ tool for counting cells, but you will have to be able to determine what you are counting... To be more specific in terms of quantification, I would use cell specific markers and flow cytometry.
  • asked a question related to Neutrophils
Question
2 answers
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?
Relevant answer
Answer
Thank you so much. That is helpful
  • asked a question related to Neutrophils
Question
1 answer
Does anyone know if there are any distinct markers for N1 and N2 neutrophils that work with flow cytometry?
Relevant answer
Answer
Dear Shiva!
Figure 1
Different role of N1 and N2 neutrophils in cancer. Neutrophils could be polarized into N1 phenotype under the
Table 2. Phenotypic features used as readout markers for the characterization of in vitro polarized neutrophils.
  • asked a question related to Neutrophils
Question
1 answer
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?
Relevant answer
Answer
Hello
Neutrophils are fragile, and about 70-80% neutrophils become apoptotic after 20 hours in culture. Experiments are usually performed on freshly isolated neutrophils rather than on those which are in culture.
How is it that you are getting <10% death after 20h in culture?
Can you share your views.
Best Wishes.
  • asked a question related to Neutrophils
Question
1 answer
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?)
Relevant answer
Answer
Dear Basak!
Yes, you can use MPO to study neutrophill-platelet aggregation
Analysis of MPO binding to human platelet membrane
Freshly washed platelets (2×108 cells/ml) were incubated with gentle shaking in a phosphate buffered saline (PBS) (10 mM Na2HPO4/KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), containing 1 mM CaCl2 and 0.5 mM MgCl2 with or without MPO for 10 minutes at 37°C. In some experiments cell suspension was supplemented with sugars (α-methyl-D-mannoside, lactose or N-acetyl-D-glucosamine at the final concentrations of 60 mM) or either with 50% embryonic calf serum or platelet-poor plasma. Then cells were applied on poly-L-lysine-coated glass slides. After 30 minutes the samples were washed with PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes, then again washed with PBS. To visualize MPO, cover slips with platelets were incubated for 1 hour in PBS containing anti-MPO antibody (1:5,000 dilution). After being washed with PBS three times to eliminate the excess of antibodies cover slips were kept for 1 hour in PBS containing anti-rat IgG-FITC antibodies (1:10,000 dilution). Then cells were again washed three times with PBS before mounting on slides. Images were acquired using laser scanning confocal microscope LSM 510 META (Carl Zeiss, Germany) with an immersion lens Plan Apochromat 63×/1.4 Oil (Carl Zeiss, Germany) and processed using LSM 510 software.
Measurement of human platelet aggregation
Platelet aggregation studies were performed using both optical and impedance aggregometry. Platelet aggregation was detected by recording changes in light transmission at 540 nm of cell suspensions at 37°C on a computerized aggregometer AP 2110 from SOLAR (Minsk, Belarus). 400 µl PRP (2.5×108 cells/ml) or washed platelets (2.5×108 cells/ml) in PBS, containing 1 mM CaCl2 and 0.5 mM MgCl2 were incubated at 37°C with stirring for 3 minutes (with or without MPO) before adding an agonist (ADP to PRP and thrombin to washed platelets). Aggregation curves were recorded for 10 minutes. To quantify agonist-induced aggregation we used the maximal rate of cell aggregation calculated automatically by aggregometer software. Electronic impedance aggregation measurements were performed using an impedance lumi-aggregometer Chrono-log 700 (USA). An aliquot of whole blood (0.5 ml) was diluted with an equal volume of prewarmed isotonic saline and incubated for 5 minutes at 37°C with or without MPO. Impedance of each sample was monitored in sequential 1-minute intervals until a stable baseline was established. Then ADP (5 µM) was added and aggregation was continuously monitored. Impedance aggregometry results are expressed as amplitude (or maximum aggregation) [ohm] at 6 minutes after reagent addition.