Immune Response - Science topic

I believe that complete RPMI-1640 (10% FBS, Pen-Strep-Gen, enriched with mercaptoethanol should do the trick. DMEM would be an option as well.

I agree with Carlos Ardavin.

Please ask you questions to ResearchGate in English.

English is the Language of Science.

Microbial communities in fish modulate immune responses by:

1. Immune System Development: Gut microbes help develop the immune system, promoting both innate and adaptive immunity.

2. Pathogen Exclusion: Beneficial microbes outcompete pathogens, preventing infections by forming protective barriers.

3. Metabolite Production: Microbial metabolites like short-chain fatty acids regulate immune responses, reducing inflammation and enhancing immune cell activity.

4. Training Immunity: Microbes can "train" the immune system, improving pathogen recognition and response.

5. Inflammation Control: Microbes balance inflammatory and anti-inflammatory responses, preventing tissue damage.

Application in Aquaculture:

1. Probiotics: Use of beneficial bacteria to enhance gut health and immunity.

2. Prebiotics: Non-digestible compounds promoting beneficial microbes.

3. Synbiotics: Combining probiotics and prebiotics to improve immune resilience.

4. Vaccination: Microbial interventions can enhance vaccine efficiency.

5. Reducing Antibiotics: Supporting microbiome health reduces reliance on antibiotics.

6. Microbiome Engineering: Tailoring species-specific microbiomes for better disease resistance.

These strategies can improve fish health, reduce disease outbreaks, and minimize antibiotic use in aquaculture.

Will you please read what I have written in the near past !

Dear Getu Habte

I found this article, that I also have added above, as I think it is highly relevant.

interested

Kindly check..

https://www.yashodahospitals.com/covid-19-faq/which-vaccine-is-effective-against-delta-variant#:~:text=COVID%2D19%20Frequently%20Asked%20Questions%3A&text=Although%20the%20current%20vaccines%20are,due%20to%20the%20Delta%20variant.

Please also see the following RG links.

Thanks a lot for your response if you have an intesresting patient we can publish a case report

Not at all . Paracetamol is safer than NSAID ( severe side effects and not to be used for ulcer , kidney pateints ) . HChQ is better than NSAID to reduce inflammation of Covid19 .

Interesting. As a teacher in contact with relatively large numbers of children it is not unusual to contract colds and flus regularly. Children appear to be great carriers of viruses but not as susceptible to them.

It is well known that older people have immunosenescence that predisposes older adults not only to SARS-COV-2 infection, but also reinfection and poor outcomes. Even the vaccines have poor response in the older people. Strategies are being developed how to tackle Immunosenescence to reduce infection risk and improve response to COVID-19 vaccine (1-4).

1.

2.

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4.

Talha Bin Emran

Also, see the link below.

Thanks!

Hi (copy from your question into discussion loop ...)

If your aim is study immunogenicity of human drugs in animal models then best way is analysis of guidances by FDA/EMA :) and total ADA (anti drug antibodies), fraction of Nab's (neutralising - high affinity antibodies) and seroconversion time. Such data could be colected after sigle dose of human protein. Sometimes pilot study is useful. Sometimes best way to analyse immunogenicity of human drug in animal model is simply analysis of drug pharmacokinetics. Then you just see what the difference is with Nab's because this fraction of antibodies increases the clearance of the drug that is administered. You can measure the most important cytokines and cells. The meaning of such work increases when your model is relevant in pharmacokinetics and pharmacodynamic meaning. Please remember that using the animal models for comparision test versus reference drug immunogenicity could be valuable (see guidelines below) but translation into humans impossible and off value. Please remember that different doses of the duman drug can induce different immune responce. For example higher dose of the drug may give lower immune responce if drug has immunosupresive action. Moreover in case of some drugs multiple dosing may decrease immune responce for example ustekinumab or infliximab ....

Some explanations from old FDA guidance could be usefull in your work (document is not available now)

„Animal immunogenicity assessments generally do not predict potential immunogenic responses to protein products in humans. However, when differences in manufacturing (e.g., impurities or excipients) between the proposed product and the reference product may result in differences in immunogenicity,

measurement of anti-protein antibody responses in animals may provide useful information relevant to patient safety. Additionally, significant differences in the immune response profile in inbred strains of mice, for example, may indicate that the proposed product and the reference product differ in one or more product attributes not captured by other analytical methods.”

GUIDANCE FOR INDUSTRY SCIENTIFIC CONSIDERATIONS IN DEMONSTRATING BIOSIMILARITY TO A REFERENCE PRODUCT FDA 2012

" A risk-based evaluation, focused on the mechanism of action of the therapeutic protein product as well as results of animal and in vitro evaluations should be performed to determine the need for collection of preand post-dose cytokine levels in the early phase of clinical development. In case of a clinical adverse event, such an evaluation may provide evidence to support the clinical diagnosis of cytokine release syndrome and help distinguish this entity from other acute drug reactions "

Guidance for Industry Immunogenicity Assessment for Therapeutic Protein Products

The immunogenicity of therapeutic proteins- what you don’t know can hurt YOU and the patient

" Therapeutic proteins show species differences in most cases. Thus, human proteins will be recognised as foreign proteins by animals. For this reason, the predictivity of non-clinical studies for evaluation of immunogenicity is considered low."

but .......

" the comparison of the antibody response to the reference product in an animal model may be part of the comparability exercise both for similar biological medicinal products "

" Qualitative or quantitative difference(s) of product-related variants (e.g. glycosylation patterns, charge variants) may affect biological functions of the biotechnology-derived protein and are expected to be evaluated by appropriate in vitro assays. These differences and impurities may have an effect on immunogenic potential and the potential to cause hypersensitivity. It is acknowledged that these effects are difficult to predict from animal studies and should be further assessed in clinical studies. "

Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues

Some nice papers about could be useful

Hope it's helpful

Best regards

Tomasz

In my personal opinion, asymptomatic Covid patients have a strong immune system, fight the virus and do not need vaccinations.

Yunus Shukor Thanks.

Indeed between a choreographed dance of hypermutations and recombination, gene splicing and translocation of gene segments to generate an enormous variety of VxDxJxC expressions for an antibody or the existence of a reverse translatase that would process the exposed epitope of an antigen and encode an RNA segment for the hyper variable segment of an antibody, nature and Occam‘s Razor I feel would pick the latter.

Thanks you M. Y. Shukor.

If you're just doing antigen recall... why do you need the whole virus? I mean technically, if you want ALL the potential viral antigens, just get an inactivated virus and use that.

Otherwise, there are a lot of recombinant proteins for SARS-CoV-2 and the other coronaviruses, and there are also overlapping peptides.

Now if you want to look at what T cell responses are during a natural infection and you don't have a BSL-3, there are psuedovirus systems out there that you can use by inserting your protein of interest.

Detection of Immunoglobulin M (IgM), IgA, and IgG Norwalk Virus-Specific Antibodies by Indirect Enzyme-Linked Immunosorbent Assay With Baculovirus-Expressed Norwalk Virus Capsid Antigen in Adult Volunteers Challenged With Norwalk Virus

Hello Dmitri,

We have evaluated the safety and toxicity profile of human embryonic kidney-derived EVs after intravenous administration in mice. Overall, we did not see toxicity effects or immunogenicity associated with our EV treatment.

Similar results were also reported by other groups (here is a nice study).

However, many factors including the EV dose, source of the cells, frequency of EV administration, administration route, etc. can have an impact on the overall toxicity of EVs. I would recommend you to perform a small safety study before your experiments to be sure that your EVs do not induce toxicity and immunogenicity in vivo. I hope this helps.

Best wishes,

Elisa

Hello Sandeep, how are you? Well, there are some reasons for this trouble. Primary, maybe your primers are not working or your cDNA samples are not well synthesized. As answered before, you can perform a conventional PCR to check if the primers are working. Also, use your endogenous control (GAPDH, ACTIN, 18s) and one sample that you know that your primers work to verify if your samples are well synthesized. Run an agarose gel and check the size of the fragment. If the gel does not appear any band, your sample are compromised or your primers are not working. Another problem is that your sample has a low quantity of your target gene, and the amplification curve is not detected by the machine even after several cycles. Usually we work with a diluted cDNA sample. The thermocycler are very sensitive to a low quantity of copies, but there is a limit to that. Maybe you need to concentrate your sample to initiate the amplification earlier. Thus, the thermocycler can detect the amplification signal. After all this, if you still detecting no CT value of your target gene, but your endogenous control is good, maybe you should consider that your target gene do not express in your sample. Good luck.

The immune response that develops in a viral infection is first given by the innate immune response. The virus enters the cell and deposits its genetic material that is recognized by endosomal TLRs (TLR3, 7, 8, 9) or by proteins of intracytoplasmic recognition (MDA-5, RIG-1 and DAI) of foreign DNA and RNA, these proteins recognizing foreign nucleic acid will activate the kinase proteins that will then activate the transcription of IRF (interferon regulation factor, IRF1,3 and 7 ) that enters the cell nucleus and activates the transcription of interferon type I (alpha and beta). This cell will produce type I interferon that comes into contact with neighboring uninfected cells by interferon I and II receptors, getting the cell into an antiviral state. This state is caused by the activation of the transmission pathways of jack-stat signals that induce the expression of genes whose products interfere with the replication of the virus in uninfected cells. We must remember that type I interferon (alpha and beta) increases MCH-I expression, activates NK cells and promotes differentiation of Th virgin lymphocytes to Th1.

I hope the information provided helps you, regards.

La respuesta inmune que se desarrolla en una infección viral se da primeramente por la respuesta inmune innata. El virus ingresa a la célula y deposita su material genético que es reconocido por los TLR endosomicos (TLR3, 7, 8, 9) o por las proteínas de reconocimiento intracitoplasmatico (MDA-5, RIG-1 y DAI) de ADN y ARN extraño, estas proteínas al reconocer ácido nucleico extraño activaran las proteínas cinasas que luego activaran la transcripción de IRF (factor de regulación del interferón, IRF1,3 y 7) que ingresara al núcleo celular y activara la transcripción de interferón de tipo I (alfa y beta). Esta célula producirá interferón de tipo I que entrará en contacto con las células vecinas no infectadas mediante los receptores de interferón I y II, consiguiendo que la célula entre en un estado antivírico. Este estado se da por la activación de las vías de transmisión de señales jack-stat que inducen la expresión de genes cuyos productos interfieren en la replicación del virus en las células no infectadas. Debemos recordar que el interferón de tipo I (alfa y beta) incrementa la expresión de MCH-I, activan las células NK y promueven la diferenciación de los linfocitos Th vírgenes a Th1.

Aluminum based adjuvants (aluminum hydroxide and aluminum phosphate) are powerful adjuvants in rodents, including mice, for protein antigens to elicit a high humoral immune response (antibodies). Selection of aluminum hydroxide or aluminum phosphate depends upon the pI of the protein. These adjuvants are cheap and you could prepare in-house. Please find attached an article describing methods to prepare these adjuvants in the lab and even in commercial scale. This article also discusses about selection of aluminum hydroxide or aluminum phosphate based on pI of the protein.

Dear Narendra Kumar Chunduri

It's the clonal selection theory of adaptive immunity, meaning that only T cells with a TCR specific for the peptide presented via the MHCII can get activated and will proliferate (if the get the correct co-stimulatory signal at the same time). If you know, which MHCII alleles your cell line expresses, you might have a chance to know, which peptide they could theoretically present and then if you have a T cell line with a TCR specific for that peptide you can set up straightforward co-stimulation assays that are outlined in the paper. If you RNA-seq data is good, you don't need to phenotype your cells for their HLA alleles as you (or a good bioinformatician) should be able to parse out, which alleles they express right away. Then you can take it from there and follow the assays outlined in the paper from Ohyama et al.

All the best & good luck with your experiments,

Michael

I think, the most important rule should be coincedence of genetic backgrounds for recipient, tumor and injected cells. They should derive from BALB/c mice. Protocols can be very different in dependence of therapeutic range of injected lymphocytes, the aggressiveness of the tumor, etc.

Please make your question in English!

Hi Dr. Al-Salhie, unfortunately your link shows an equipment that is called "Crocodile" ( the commercial name of the instrument is crocodile, a device to pipette, wash and incubate plates only). However, Berthold technologies does not sell ELISA assays directed to crocodilian proteins, just as nobody else does for reptiles. Believe me, I searched everywhere for antibodies against snake proteins.

Thank you though, for a split second my hopes were high...

See this link may be helpful

Regards

See this link of article is useful

Good luck

agree with dr Torsten Goldmann

Hello

This is an interesting topic.

Zn is useful in the body in very small amounts and ZnO helps to enrich the blood with oxygen. This makes it possible to enrich the regeneration of red blood cells. The remaining Zn is excreted through the syringe system or accumulates in the bones if it is in increased doses.

Guy Dutau,  thank you for your response

works fine, but everything depends on the exosome dose (make sure its physiological) and purity (make sure there are no admixtures in your prep- eg trace amounts of chemicals)

Hi Keerit,

here are some suggestions.

For T cell identification (CD3 or TCRb plus CD4 and CD8); subsequently activation markers include: CD44 (upregulated during persistent T cells activation), CD62L (naive T cell marker that's inversely downregulated), CD69 (upregulated intermittent T cell activation), and possibly CD25 (traditionally a Treg marker, but also expressed on activated T cells)

For B cell identification (CD19 and/or B220); subsequently activation markers include: CD54 (ICAM1), MHCII, CD80, CD86, surface IgG

For proinflammatory Macrophage identification (CD11b and/or F4/80); subsequently activation markers include: MHCII, ICAM1, iNOS and NADPH oxidase subunits like i.e. p47phox

Most of the above mentioned markers are on the surface and therefore detectable with a simple extracellular staining. You can of course add, intracellular cytokine/ transcription factor stainings for TH1/ TH2/ TH17/ Treg differentiation. I'd particularly have a look at TH1 and Treg stains for example with IFNg and FOXP3. Moreover, you can give a thought to a perforin and granzyme B staining on T cells, which may particularily be suitable for extrapolating the likelihood of transplant rejection.

Hope that helps!

Good luck with your experiment!

Michael

Heat-killed bacteria can be stored at -20.

Compare to purified bacterial ligands (LPS, PAM3CSK3, etc) you can expect more variations in your experiments using HK bacts. So if you want to have a robust stimulations you are better of using specific bacterial (TLR) ligands.

Try invivogen website :)

Interesting topic.  As is typical, the answer will depend on your research question.  It isn't clear to me whether the cases in your study are of TB disease or latent TB, and if they are co-infected with helminths.  The strongest risk factor for all of these is probably foreign birth, so if you select controls from the general population you will likely find this to be the strongest association in your study.  So you may want to match on that if it is not a variable of interest.  Age is less strong for TB but still important so matching might be important.    

If you are interested in studying whether factors, such as co-infection, might lead to progression of latent TB to TB disease, then you might want to select controls who have LTBI.  This would probably obviate the need for matching on foreign birth or age.

If you simply want to see the differences in immune responses between co-infected vs mono-infected, you might want to compare 4 groups: uninfected for both, infected for both, and mono-infected TB and helminth.  In that case, I would think that matching would be related to those variables which are known to affect your immunological outcomes of interest, which will depend on which outcome you select.  I would think about age and foreign birth as potentially important sources of confounding, but also comorbidities and other factors.

Dear Donna, yes, the protocols are very similar. I titrate serum IgGs, and do not have experience with your sample types. Nevertheless: Some considerations that might give you a clue about the source of your problems:

- How do you prepare the serial dilution of your samples? Do you use a too high dilution factor that might be responsible for the sudden signal drop?

- Do you observe the same problem for all of your sample types (caecum, colon and faecal samples)?

- Do you have a positive control to check whether the current protocol works for IgA?

- Did you include a negative control to exclude other problems?

- Have you tried higher BSA concentrations for blocking to decrease unspecific binding?

Thanks for the additional comments! Unfortunately I don't think these approaches would be specific enough, because I'm talking about tissue sections and the mentioned downstream signaling molecules are not exclusively used by TLRs, so their activation doesn't prove TLR activation.

KLH is a transporter that allows the immune system to function when the molecule of interest with which it is associated is too small.

This is particularly the case with most synthetic peptides which possess too few distinct epitopes to be taken up by dendritic cells on the one hand, then CD4 T cells and finally by memory B cells. It takes two to three separate epitopes to have a good immune response. KHL will then lend one or two epitopes to ensure an effective immune response.

In addition, KLH can be used to purify the target-KLH chimera, and use the residual anti-KLH response to follow the immune response.

The other carriers that can be used are either immunostimulants (Al3 +, TLR...) Or presentation particles (VLP, micelles, liposomes, nanoparticles, etc.)

you may couple your peptide to a carrier protein such as ovalbumin or KLH, but take care to end up with a balanced molar ratio of peptide to carrier, for example 2 or 3 peptide molecules per carrier molecule

In reality there are only articles that tend to show that seroconversion goes hand in hand with protection.

But the great majority of the works carried out all over the world lead to the opposite: an immune response giving no protection.

But these works are never published, without interest! What's the point!

For example: Influenza, HIV, Dengue, CMV, Leptospira, Pseudomonas, cancer cells ....

I suggest that you read a textbook chapter  on hapten -carriers and why they are important for the interaction of T and B cells!

Hello,

Fixing the cells, immunofluorescent staining with specific antibody and fluorescence microscopy. The protocol optimized for your cells and protein of interest is very important, it should be searchable.

Protein Sciences sells rHA from several viruses, including HK/156/97.  http://www.proteinsciences.com/

Hi Qingrong

Look PDF attached may help you.

Good luck

They are handled by Kerafast.  If you check their website, they are available.

Your question is very broad. To give you some hints: it all depends on the GMO and which antibiotic you are interested in.

ADCC would work if you had an antibody against a component of the virus that shows up on the membrane of the infected cell.  but would not work (that i know of) if the antibody were against a molecule exposed on the virion, but not on the cell.  still.......in a mixed antibody response, there should be some antibodies that work via NK cells and ADCC.

Dear Nicholas Nii-Trebi,

Please have a closer look to:

Best regards,

Thomas

If you are looking to do RNA-sequencing analysis to get gene expression profiling, but do not have experience yet with bioinformatics, I would suggest using the Galaxy server:

You can find some tutorials here: https://github.com/nekrut/galaxy/wiki

-Benjamin

Dear Collegue

I have colleague ... Dr  Rina Triasih Indonesian Pediatricians from Universitas Gadjah Mada .. PhD graduate from Melbourne University and published many international publication. If you need more information about her I will give you further contact information

Best wishes

Awalia Febriana 

it would help to know what you're stimulating and measuring as "immune response".  in the meantime here is a possibility.  when you use SiRNA, you're likely using something like lipofectin to get it into the cell?  that perturbs the membrane and perhaps stimulates the inflammasome.  so the cell gets at least partially activated.  if you're comparing the SiRNA to an empty vector, then the empty vector is also causing partial activation and the differences between control and experimental will be small.  when you use ShRNA, you wait for much longer to get a stable line, so the membrane perturbation will have settled down and your cells will be more in a "resting" state.

in any case, could you offer some more detail as to what you're doing?

Please refer this paper

Many thanks Willem. After some more analysis it looks like the time lags may also have something to do with different routes to infection between males and females, however, the differential immune responses are of vital importance.

Dear sir,

See this link may be useful for you.

Hepatitis C coinfection enhances sensitization of CD4(+) T ...

www.ncbi.nlm.nih.gov/pubmed/22024520Traduire cette page

de C Körner - ‎2011 - ‎Cité 18 fois - ‎Autres articles... increased rates of CD4(+) T-cell apoptosis in HCV-infected HIV-positive patients ... Susceptibility to FasL-induced apoptosis was analysed by incubating isolated ... CONCLUSIONS: Our findings suggest a synergistic mechanism in HIV/HCV ...

Well the whole point of using k.o. mice to see the phenotype of a specific gene on different biological situations. It is higly depends on the qestion you ask. There are some specific models of spontanious desease development due to gene k.o. yet again you shuld consdider what is the scientific qestion you adressing with this. 

Thank you Sir Yuksel...but why Balb/c any specific reason?

Take a look at this review, it might help you.

Dear Andres,

The following covers the answer to your question:

FEMS Immunol Med Microbiol. Author manuscript; available in PMC 2015 May 11.

Published in final edited form as:

FEMS Immunol Med Microbiol. 2012 Dec; 66(3): 436–444.

doi: 10.1111/1574-695X.12010

PMCID: PMC4426994

NIHMSID: NIHMS413557

Pluronic P85 enhances the efficacy of outer membrane vesicles as a subunit vaccine against Brucella melitensis challenge in mice

Neeta Jain-Gupta,1 Araceli Contreras-Rodriguez,2 Ramesh Vemulapalli,3 Sharon G. Witonsky,4 Stephen M. Boyle,1 and Nammalwar Sriranganathan1,*

Abstract

Brucellosis is the most common zoonotic disease worldwide and there is no vaccine for human use. Brucella melitensis Rev1, a live attenuated strain, is the commercial vaccine for small ruminants to prevent B. melitensis infections but has been associated with abortions in animals. Moreover, strain Rev1 is known to cause disease in humans and cannot be used for human vaccination. Outer membrane vesicles (OMVs) obtained from B. melitensis have been shown to provide protection similar to strain Rev1 in mice against B. melitensis challenge. In the present work we tested the efficacy of Pluronic P85 as an adjuvant to enhance the efficacy of Brucella OMVs as a vaccine. P85 enhanced the in vitro secretion of TNF-α by macrophages induced with OMVs and P85. Further, P85 enhanced the protection provided by OMVs against B. melitensischallenge. This enhanced protection was associated with higher total IgG antibody production but not increased IFN-γ or IL-4 cytokine levels. Moreover, P85 alone provided significantly better clearance of B. melitensis compared to saline vaccinated mice. Further studies are warranted to find the mechanism of action of P85 that provides non-specific protection and enhances the efficacy of OMVs as a vaccine against B. melitensis.

To view the full paper, please see attached file.

Hoping this will be helpful,

Rafik

Correct! 75% survival

Hi, just a short thought: Helminths usually initiates a Th2 type immune response. The C57Bl/6 strain is not a very good Th2 responder strain in comparison to other inbred strains such as Balb/cJ or NIH/Ola, which I have also worked with in allergy studies. (Allergy is also Th2 response). For example - I used the Th2-promoting adjuvant aluminiumhydroxide, but the antibody response was much higher in the two latter strains compared to the C57Bl/6-strain. So as Frederic Beaudoin points to, it is very important to consider the type of immune response you want to study and from this select strain and adjuvant. Best regards, Jitka

you can't.  the immune response in vivo will be the result of all sorts of signals that don't occur in vitro.  so it really depends on what you want to know.  if the strength of the response is all you need, then, as listed above, the mixed lymphocyte culture can give you a reasonable idea.  but if you want to know more than that (like what kind of response occurs to different transplants, or the kinetics of the response, or data on the homing of cells to the target organ, or what types of cells are involved in the rejection or tolerance, then the Mixed lymphocyte reaction is not enough.  So, it would be helpful to know what question you are asking.

It would be helpful to know which reporter mice you have available, and which parasite you're studying; if this is just a general question about reporter mice or a particular set.  there are so many that without a few more details, this is a difficult question to answer.

For example, if you wanted to enumerate the T cells responding to flu, and you didn't care what kind of response they make, you could make a reporter mouse whose T cells light up when they activate CD69.  a floxed stop on the reporter color, driven by cre on a CD69 promoter would permanently color the responding cells, whereas a construct with the color on the CD69 promoter would color them temporarily.

If you wanted to know what kind of responses a particular parasite drives, then you could use reporter mice whose cells become colored when they make particular cytokines.  or you could simply look at the classes of antibodies the mouse makes (without having to use a reporter mouse). 

So it really depends on what question you're asking

There will also be a lot of TNF produced!

Fabulous question. I thought one of the reasons we avoid administering pharmocological antipyretics was their anti-inflammatory effects not necessarily the effect of reducing core temperature. CVVHDF cools by convection so the effect of cooling is confined to the reduction in temperature rather than the effect on of reduced cytokine release on the inflammatory response. 

I will continue to follow this question with interest. 

You may need to do some optimization as suggested above. Titrate the PMA/ionomycin and also do a time course. Calcium flux should happen quickly- you may be missing the peak by waiting 30 minutes to run the samples. We look within a minute.

Hi, it could be several things-buffer pHs (most likely), unhealthy cells, IgE or antigen not good / wrong concentration, lysis detergent wrong concentration. We use Tyrode's buffer pH 7.4, substrate=4.5, and stop solution=10.7. Even if you made the buffers at the correct concentration, the Tyrode's buffer pH could change over time, even with a buffering agent, so I recommend checking the pH before every experiment. Generally 1ug/mL IgE and 100ng/mL DNP-BSA are good, although the valency (number of DNPs per BSA molecule) could change these figures. 1% v/v Triton-X is good for lysis. Generally 1 hour stimulation is good and consistent. You may see some morphological changes, but don't expect to see complete degranulation using IgE and antigen. If you're using SPE-7 IgE, be careful as it can activate cells without antigen.

Hi, Sharon,

Yes, they are differentiated into macrophages with PMA before infection. I do not think that using inactivated/dead parasites is the best shot in this kind of analysis, because even in these conditions, the antigens are still there, and they can trigger an immune response and probably will alter miRNA expression. Thus, it would not be a negative control anymore.