Science topic

Cancer Immunology - Science topic

Cancer Immunology are breakpoints in tumor immunology, cancer immunospecific theraputic targeting, adjuvant immunotherapy
Questions related to Cancer Immunology
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I have been finding the proof that this epitope called NLVPMVATV does not induce cytokine storm for days, but in vain. Could anyone please help me? I really need a reference paper to support this or else my project will be in a great trouble.
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Hi,
Check the references below:
HCMV UL83/pp65 (NLVPMVATV)
CMV pp65 peptide NLVPMVATV (HLA-A*0201) is a single peptide for stimulation of T cells. The peptide from human cytomegalovirus (CMV) phosphoprotein (pp65) is synthesized as it is presented by the MHC class I HLA-A*0201 allele and can be used for targeted in vitro generation and expansion of antigen-specific CD8+ T cells.
Reference:
"Although the anti-pp65 antibodies and the pp65 antigens are detected in immune-depressed patients with active viral infection [16], the antibody response to the pp65 antigen in normal infected individuals is not always detectable by immunoblotting [17]. It has been reported that the pp65 antigen is targeted by a cell-mediated immune response and that its vaccination can induce a pp65-specific CTL response"
Reference:
Hope it helps,
Best regards
Tomasz
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I saw many papers are introducing or immunizing mice with ovalbumin (OVA), will this induce a cytokine storm? Is there any paper suggesting that OVA doesn't induce cytokine storm? I've been searching this for days, please help.
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No, there are essentially 3 major types of immune response to infection: an antiviral response which illicits interferon, a pro inflammatory response that makes il1, il6, tnfalpha etc wich is normally directed against bacterial and fungal infections ( and can lead to cytokine storm) and then the allergic response directed against allergens like ova that generates il4, il10, il13.
In mouse models, balb/c mice are typically used to study allergic responses as thy are inherently more allergic and c57bl/6 are used to study proinflammatory responses and diseases because they are inherently more prone to this type of immune reaction.
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My experiment consists of a ex vivo stimulation experiment on isolated human monocytes.
Each biological replicate consists of the same isolate of human monocytes (derived from the same donor) and for each biological replicate there are three treatment conditions. The dependent variable for the experiment is secretion of a specific cytokine. In the analysis I am comparing the mean cytokine secretion of each group against every other group.
I am however unsure when comparing these means whether the data points for the three treatment conditions from each biological replicate should be considered paired or unpaired, as this will obviously influence the statistical test I use?
Thanks,
Gabriel
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As far as I understand, your data points/conditions are nested within individuals (donors) creating non-independence. In other words, the same individuals provide data for each condition. If that is true, then a paired test such as repeated measures ANOVA would be most appropriate. One way to examine this is by looking at whether the observations across conditions are correlated. For paired observations, you will typically find a positive correlation of the DV scores across conditions reflecting the non-independence of the data points.
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Hello.
CIBERSORT or CIBERSORTx calculates "p-value" for each analyzed sample. This value seems to suggest the reliability of CIBERSORT analysis for each sample. If it will be p>=0.05, we may exclude the sample from the downstream analysis. However, sometimes we experience that more than half of our samples are excluded by the p-value. It will make the downstream analysis difficult.
Does anyone have a similar experience, and how do you handle this issue?
I am looking forward to your valuable advice.
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Sorry it is outside my aera of expertise
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Bispecific antibodies, trispecifics or other formats have been and are still explored as powerful drugs against various cancer types. However, potentially these therapeutic agents could have a much broader application range. What are the reasons, researcher focus on cancer (beyond the obvious ones like incidence rate, death toll...)? The concept of bridging two or more epitopes on different cell types could in my mind potentially be extended to quite a vast range of targets. What about engaging bacterial cells? And what about non-peptide epitopes? Curious to hear some interesting thoughts!
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Hi,
Very important question. Moreover, the question is why (.... from my current knowledge please correct if I'm wrong) from more less 100 mAbs approved by FDA only 2 are bispecific ..... I think another trend is competitive for the "multispecificity" of mAbs. Its cocktails of mAbs (cocktails therapy). See some examples below:
1) atoltivimab + maftivimab + odesivimab-ebgn
2) bamlanivimab + etesevimab
3) IgG1(M777-16-3) + IgG2(62-71-3)
4) bamlanivimab + etesevimab
I think that cocktails therapy will be a more dynamic trend than "multispecificity" (because of higher and higher availability of different monospecific monoclonals)... and many other reasons,
Best regards
Tomasz
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I have the sequence of a mouse antibody and want to know which are the closest related human VH sequences by percentage of similarity or alignment to my murine VH sequence. I found all the IGHV repertoire but I'm kind of new using IMGT and what to know if someone out there more experienced than me knows how to blast a mouse VH against all the repertoire of human VH contained in the IMGT.
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Interested
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Hi,
I want to make the IsoFlow Sheath liquide in our lab by following the chemical composition given by the company ( mentioned down below) , i want to know if any of you tried this before and if it works without any risk during the aquisition process. and i would like to know also if there are any tips or precautions to take when preparing the solution.
Chemical composition for 10L :
Sodium Chloride......................7.93 g/L
Disodium EDTA........................0.38 g/L
Potassium Chloride.................0.40 g/L
Monosodium Phosphate.........0.19 g/L
Disodium Phosphate..............1.95 g/L
Sodium Fluoride......................0.30 g/L
Kind regards.
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IsoFlow in Beckman coulter or sheath fluid in BD machine are the isotonic buffers solutions which may be replaced by Autoclaved and filter sterilized PBS.
You should be extra cautious while using in-house sheath buffer as any particle or growth may block your tubes in flowcytometer.
We have used Autoclaved and filter sterilized PBS in BD instrument and it never caused any issue.
Once you use any in-house buffer, the parent company will consider it void of warranty. So, never let the company people know that your are not using their isoflow.
Good luck,
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I am currently finalizing my Master of Science in Biochemistry. I am interested to pursue my career in Cancer Immunology. Meanwhile reading research papers, I would love it if my reading gets one step ahead. Any small or big advice would be very helpful to me!
Thank you for taking out time to read my post :)
Nikita Maurya
Master of Science in Biochemistry
K.J.Somaiya College of Science and Commerce
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Go to the web page of your journal of interest and register as a reviewer
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Please find attached the full medical file of our patient and all biological reseaults included.
We are seeking to explain how can she produce such levels of immunoglobulins with no B cells expression.
NB ! : * The patient ddidn't recieve any immunoglobulins injection.
* We made another dosage for immunoglobulins levels after 01 moth of the first one and we had the same resault ( high levels ).
Best regards
Msc, Abdelwahab
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First thing i would like to comment on it that you may change the clone of CD20, check CD19 as well if they are even few?
Secondary, The absence B cells might be the drug effect (Like: Rituximab or any alternative/traditional medicine).
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In my current project I am planning to isolate mononuclear cells from peritoneal ascites of cancer patients using ficoll centrifugation. However, following isolation of the mononuclear cell layer, I would still be keen to freeze the remaining cells in the pellet (tumour cells, stromal cells, granulocytes etc). Is there any problem with resuspending the pellet, washing the Ficoll off, lysing RBCs and then freezing?
Thanks,
Gabriel
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I see that Alexandr and Maria have botj given a recipe for freeze medium above and i would like to add that post-thaw viability is increased if the freeze medium also has FBS.
Our recipe for freeze media is
- RMPI (supplemented with 1% antibiotic/antimycotic solution)
- 20% FBS (10% is fine though)
- 10% DMSO.
Anyway, i have seen each laboratory has it's own recipe so as long as it makes you happy, it's fine.
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Oncology is a branch of medicine that deals with the prevention, diagnosis, and treatment of cancer. A medical professional who practices oncology is an oncologist. The name's etymological origin is the Greek word ὄγκος (óngkos), meaning 1. "burden, volume, mass" and 2. "barb", and the Greek word λόγος (logos), meaning "study".
Cancer survival has improved due to three main components: improved prevention efforts to reduce exposure to risk factors (e.g., tobacco smoking and alcohol consumption), improved screening of several cancers (allowing for earlier diagnosis), and improvements in treatment.
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Lauren Pecorino
Molecular Biology of Cancer: Mechanisms, Targets, and Therapeutics
3rd Edición
ISBN-13: 978-0199577170, ISBN-10: 019957717X
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The event of death by metastasis or recurrence is very common, many researchers have linked the tendency of some tumors to re-appear to the presence of occult or dormant cancer cells that may have a phenotype that allows them to remain "hidden" from the immune response. However, the understanding of these cells and the mechanisms that they use to achieve evasion remain mostly unknown. It is critical to understand these cells better and to be able to detect their presence for they are of great therapeutic importance.
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Hi
LGR5 is the best biomarker for detection of cancer stem cells in colorectal patients .
u can browse our my profile and see our paper about that
Regards
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When doing T-cells activation assay, for example looking for CD25 ,CD69 expression or ELISA for cytokine release, is there a difference between the coated antibody or coated protein stimulation and cell stimulation using cell lines? What is guide to use the stimulus.?
Thanks,
Abdul
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Short answer, yes there is. The first two is almost always global, with the last one... with the potential of being more specific.
For coated antibodies, it is almost always CD3/CD28, and the coating is for crosslinking. Otherwise, it doesn't work.
As for protein... do you mean an mitogen? Like ConA? or a cytokine like IFNg? Either way, the activation pathways are different and for these, there is no need to coat the plate. They will work in suspension.
Cell stimulation using cell lines...Unless you are looking at stimulation using unmatched HLAs...Most of this is using cell lines preloaded with a specific antigen or antigenic peptide.
If you can give more information about your goal... we can give you an idea of what would be the best way to go about it.
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Hello.
I work in a cancer immunology group. We need to deplete CD45 (.1 /.2) OT-1 cells previously transferred to CD45 (.1) mice, with aCD45.2 depletion antibody. But we have doubts about the dose to use. We usually deplete CD8+ T cells with 60ug of aCD8 depletion antibody (YTS169.4) but this time we need to deplete a smaller number of cells and maybe 60ug of aCD45.2 is an excess. In their work, Laura Mackay's team uses about 10ug of the antibody aThy1.1 (HIS57) to deplete CD8+ T cells Thy1.1+.
Beforehand thank you very much.
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I am so sorry for such a delay, I had not reviewed Researgate in many weeks.
Could you solve your problems ?. We have performed similar experiments and we have not had that problem, so I am not sure what it could be. One question, does your cell therapy promote tumor eradication?
I have seen that some researchers use treatments with IL-7 / IL-15 to favor memory phenotypes that last over time favoring the antitumor response.
It could also be that your B16 cells have lost the expression of OVA, do they have a probe to follow the expression of OVA ?.
I stay tuned in case you need help.
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Hi everyone,
I would like to measure the amount of intracellular cholesterol in CD8+ T cells isolated from tumors . To do so I would like to use filipin III (Cayman Cholesterol cell based detection assay kit) to stain for cholesterol, however I am unsure if I can stain Filipin III as well as KI67.
Below is my current protocol for Filipin III staining. I would like some feedback on it, and where I could possibly add the KI67 staining step.
Resuspend isolated TILS in FACS buffer (0.5 % BSA in PBS)
Incubate for 10 mins with FC Block
Stain for surface markers for 20 minutes
Fix cells for 20 minutes with Cytofix/Cytoperm
Wash cells with FACS buffer
Stained with Filipin III for 50 minutes
Analyze promptly by Flow cytometry
Thanks !
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Hi Dominique,
As far as I can tell, it should work to simply add your Ki67 as a separate staining step following permeabilization. Otherwise, your protocol looks fine to me.
Good luck,
Max
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I need to find out if my protein has binding sites available for intermolecular interaction with other proteins. 
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Hello
CASTp, Autodock vina, Swissmodel, Qsite, Chimera, RasMol or JMOL, MOE, BIOVIA Discovery Studio.
Check in these links. It might help you.
Good Luck.
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I am doing a pilot study to assess the cytotoxic abilities of different T lymphocyte populations when cocultured with B16 Ova In Vitro. I have seen different articles labeling their effector/cytotoxic T Cells with a target dye to gate them out and look at Annexin and PI positive cells. But I do not have any target dye to label my cells. Instead can harvest my cocultured tumor + T cells and first do a CD4 CD8 surface staining with antibodies and then follow Annexin PI staining protocol? I understand the 2 stainings are different but will that affect my apoptosis results in any way? Has anyone done this before and has had luck with it? Please help me to understand better. Thanks
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Hi Sarah Ratkovich-Gonzalez , I remember that you shoul perform the surface staining first and then stain with AV (and 7AAD for example). That is because for AV staining you need to you a "special" binding buffer.
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Cancer immunotherapy comprises a variety of treatment options like, the use of different kinds of vaccines. Could the RG users explain to me the current advances on the use of vaccines in cancer immunotherapy?
Thanks,
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This article may be useful:
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In this article fig. 1. The Author has made a decision tree with 24 nodes. Each node is specified for a specific cancer tissue of origin and the couple of MicroRNA which can identify these cancer tissues of origin. My question is, if I isolate miRNAs at the node14(hsa-miR-21, let-7e), node21(hsa-miR-205, 152), node24 (hsa-miR182, 34a, 148), node10(hsa-miR-194, 382, 210), will it be enough to identify cancerous tissue originated from the lung.
Why am I asking this question, because, I want to identify cancerous tissue, which has migrated to different region but originated in the lung. So if I take miRNAs from those specified nodes, will it be enough to identify lung cancer tissue, which has migrated to different region but originated in the lung.
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microRNAs have to provide potential signature model for various cancers and other diseases. While miRNA in terms of sequencing is difficult , though being done provides huge number of miRNA in a specific cancer as you have exemplified some in your discussion. Few important learning points:
1- Specific sets of miRNAs usually express or otherwise together and they are termed miRNA families.
2-Some miRNA have the potential to act as pan cancer biomarkers but still no specificity is provided.
3-Some biomarkers are like positive or negative acute phase reactant and may rise or fall with non-cancerous disease.
4-There is some but as i experienced little correlation between blood and tissue based miRNAs
5- The science of miRNAs is still emerging and a lot more has to be learnt i guess before they are available, if available for clinical use.
6-Also need to have complete data about pre, pri and miRNA and he cleaving proteins like DROSHA, DICER and factors incorporated in RISC complex.
The whole picture is yet to appear and but hope is there that someday they may be appearing as both for diagnostic use and therapeutic targets like Riversin (spelling ?) for treating hepatitis C.
So potential is there but more research is needed to quantify and deal associated aspects of miRNA
Sorry, that I could not help you straight as i interpret the knowledge about this subject is still evolving.
Kind regards
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I am still trying to figure out on how to gate lymphocytes population on FLowJo. Attached are 5 plots where all samples are murine spleen and lymph nodes cultured in vitro.
1) My 1st question is based on plots 1,2 and 3 attached here. I am not sure where to place the gates for lymphocytes and live dead population. I was always under the impression that lymphocytes will be from 50k to 200k and live dead within 10^3 on the X-axis. As you can see from the 3 plots, if I change the gate positions I do not see a double population for CD4. Kindly help me in understanding better.
2) My 2nd question is pertaining to plots 4 and 5. Here I am seeing 2 populations on the FSC vs SSC and what I gated on are the lymphocytes I believe. My live dead population here seems a little different. I am not sure if I am actually seeing Live population as it seems really spread out. Here also if I gate my live population within 10^3, I feel my CD4 vs CD69 background is reduced. I am kind of lost with both the analyses. I would really appreciate help with this matter.
Thank you
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Hi
The most researchers used 50µM of ex-527 for treatment of cells. How can they get to this concentration, whereas IC50 of this drug is 89nM ?
I want to use this drug for Jurkat cells and molt 4. . How can I find the best concentration of ex-527 that inhibit SIRT1?!
MTS assay is good?!
No article exist setup this drug for Jurkat and molt4.
Thanks in advance
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IC50 for a compound/inhibitor for a cell line depends on several factors, including the cell line in question, solvent for the inhibitor, culture conditions, and proposed endpoint for the treatment. Therefore, it needs to be determined empirically under the desired conditions for the proposed endpoint (when known). If a study has used much higher concentration of the inhibitor for a similar endpoint, it could be due to due to other variables (cell line and culture conditions etc). It is always better to find out an optimum IC50 before performing an experiment.
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I am curious to find more about this
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Aceria pongamiae a species of mite causes gall formation on the leaves of Millettia pinnata (= Pongamia pinnata).
Please have a look at these links and PDF attachment.
Good luck!
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Actually we tried to develop a xenograft metastatic model of melanoma with NOD-SCID mice after ic injection of A375 metastatic melanoma cell line. After 4 wks by ic injection we only found oligometastatic disease, prevalently spread to bones or muscles. 
Are NOD-SCID mice suitable for this model?
Many other groups used Nude mice for the same purpose, however with different cell lines. We will soon repeat experiments with other cell lines.
Does anyone have experience with both NOD-SCID and Nude mice xenograft models of metastatic melanoma?
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We use NOD/SCID mice in our commercial A375 xenografts (http://altogenlabs.com/xenograft-models/melanoma-xenograft/a375-xenograft-model/) and they tend to work well, with stable growth curves as well. I would make sure your starting injection is done properly (we use matrigel with 1 million cells) and then monitor for any kinds of symptoms that wouldn't correspond with normal tumor growth.
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I am a graduate student and would like to know which areas have promising potential for future funding. This information would help me choose professors. Thank you.
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Samrat Das Thank you for our response Mr. Das. More specifically I wanted to know about Caner and Immunology, and which areas of therapeutics would be more beneficial for the future.
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We are currently having a hard time getting TAMs (especially Multiple Myeloma associated macrophages) from peripheral blood monocytes. Do you have any suggestions or possible protocols for doing so?
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Hi Zoabi, I am wondering about how to extract myeloma-associated macrophages directly from the bone marrow, and I saw that you had posted this similar question a while ago. Did you find an answer to this? Best, Kristian
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Our research group have developed a novel polysaccharide adjuvant we call Advax which has proved effective in multiple animal models and is also in late stage human development. Its major virtue is that it is non-inflammatory and thereby nonreactogenic.We are always looking for collaborators who might want to test it in their particular animal model or who want a GMP adjuvant for human trials. Data from each new model or application gives us additional insights into the action of this unique adjuvant.
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Following
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Tumour specificity of the immune response resides in the recognition of specific or associated tumour antigens. How the immune system do this?
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There are multiple mechanisms that the tumor uses in order to escape immunosurveillance. One of the more important ones is the acidity of the extracellular mathrix. This is mainly due to proton extrusion. Low pH decreases the activity of macrophages and T cell lymphocytes and impedes the immunologic atack on cancer.
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Dear Researchers Working in field of Tumor immunotherapy,
I am working with MC-38 colon cancer models. I had faced a critical issue when I tried to use TAA immunotherapy.
I have observed this phenomenon twice:
To study the TH1 specific response we are considering IFNγ and TNFα, When I am culturing splenocytes of tumor-bearing mice overnight with Antigen, I found there is a loss of CD8 cells. Is it very common phenomenon or I am making some mistake?
Awaiting for your crucial comments
Thanking you
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Hello,
I worked on invariant natural killer T cells, and I found that these cells become anergic upon activation with a specific antigen and they down regulate their specific receptor. you may have the same situation with CD8 cells (anergy + down regulating their receptors). And this is not the case when isolating healthy NKT cells.
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T-cells with self-reactive T-cell receptors are eliminated via the process of central and peripheral tolerance. Self-peptides are transported from different tissues in the body to the thymus, where they are presented to T-cells, which then receive pro-apoptotic signals. But how does the body know which peptides are self-peptides? Why aren't e.g. cancer neoantigens presented in the thymus as self-peptides? Is there a temporal component to it? 
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Hi Anne and Christoph,
Thank you so much your comments and references to literature, I found it very helpful!!
Kind Regards,
Martyna
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I am using a C-18 RP column - 250mm x 4.6 (id); 5micron column to separate imatinib mesylate. The stock is prepared in DMSO and the standards are prepared in the mobile phase.
The mobile phase is composed of 25mM potassium dihydrogen phosphate and acetonitrile. I have tried this MP composition in different combinations from 30:70 ACN:Buffer till 70:30 Buffer: ACN at a flow rate of 1ml/min. Additionally, I have also carried out the separation at pH 2.5, 4.5 and 5.5.
From these expts, I found that pH 2.5, MP 35:65; ACN: buffer was of a relatively better separation, as I could distinguish between DMSO and the drug peaks. 
The problem however, is that there is still peak tailing. What changes can I incorporate now?
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You have NOT changed the K prime (Capacity Factor) at all. You reduced the flow to half of what it was (from 1.0 ml/min to 0.5 ml/min). Your T zero time has now doubled from what it was before. Your previous T0 was ~ 2.9 minutes with a peak showing NO RETENTION at 3 minutes. Now your flow rate is 0.5 ml/min and the new T0 time is ~ 5.8 minutes with your peak continuing to show NO RETENTION at all (as we would predict) at ~ 6 minutes.
Previously, you stated that 30% ACN resulted in no retention. Yet you continue to try mobile phases with even more ACN on a RP column. Again, you would want to try mobile phase mixtures with less ACN (or MeOH mixes or just buffer) to try and retain the sample first.
I understand that you are brand new to chromatography, and apologize for sounding harsh, but you still have not taken the time to read the linked articles I offered, follow my suggestions or demonstrate any basic understanding of the fundamentals of HPLC at this point. Note: Adjusting the flow rate does not change the peak's capacity factor (it is still close to zero). Reducing the flow rate simply slows down the entire chromatography process. Your peak took twice as long to come off the column (because the column volume does not change) as before because you reduced the flow rate by a factor of two. Please change the method and most importantly of all, get some local help in learning the basic fundamentals of HPLC before you start to run samples. You will greatly benefit from some basic training at this point so you can begin to understand what you are doing. Randomly making changes is not the best way to learn. Experimenting without understanding the basic concepts about what you are doing is both unsafe and unscientific. Please get help before continuing.
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I want to set up an assay with PBMCs to monitor NK cells mediated lysis of cancer cells. The assay is intended for both small molecule activation of NK cells in PBMCs as well as ADCC. I'm looking for an immunomodulator drug as a positive control, e.g. Lenalidomide. However, literaure shows that activation is only 2-fold by lenalidomide (with IL-2). How can one boost this activity? Would Lenalidomide +IL-2 + IL-15 be a good combination? Or are there other agents/drugs that can specifically activate NK cells and be considered as a positive control?
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To boost ADCC or direct lysis with NK you can use IL-2 and/or IL-15. But if you want only a positive control of the NK lysis you have to use a good target for NK cells such as Daudi cell line in the presence of IL-2.
If you want to produce cytokines (IFNg or TNFa) by NK, you can use IL-12 + IL-18
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Does anyone have any experience with RET immunohistochemistry? I am interested in staining nerves in skin. Thank you
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Hello,
I recommend an antibody database available at labome.com. For antibodies to RET for IHC you can use the following link: https://www.labome.com/review/gene/human/RET-antibody.html. Epitomics has RET antibody (Epitomics, 3454-1) used in immunohistochemistry. Also, Abcam offers RET antibody (Abcam, ab1840) was used in immunohistochemistry - paraffin section on human samples (Ding et al, Exp Mol Pathol. 2013). In addition, R and D Systems provides RET antibody (R&D systems, MAB718) used in immunocytochemistry on human samples (Fattahi et al., Nature 2016). You can check the references and the description of these and other RET antibodies following the links.
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We already tried one poly- and one monoclonal GATA2-Ab which were quite unspecific.
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Hello,
I recommend an antibody database available at labome.com. For antibodies to GATA2 for WB you can use the following link: https://www.labome.com/gene/human/GATA-2-antibody.html. In particular, Santa Cruz Biotechnology has mouse monoclonal (CG2-96) for WB. Also, Proteintech Group offers GATA-2 ab (catalog: 11103-1-AP Proteintech Group) for WB. Invitrogen provides GATA-2 ab (catalog: PA1-100). Also, there are GATA-2 antibodies from GeneTex (catalog: GTX113441), Abnova (catalog: H00002624-D01P),  US Biological (catalog: G2021-40), Novus Biologicals (catalog: NBP1-82581), LifeSpan Biosciences (catalog: LS-C31147) and OriGene (catalog: TA312431) suitable for WB. You can check the references and the description of these and other GATA-2 antibodies following the links.
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I wonder wether there are shared TCRB-amino acid sequences in tumor bearing C57Bl/6J mice. We performed an immunoSEQ assay and found no overlap between our mice regarding the TCR repertoire of TILs. Does anyone have made similar observations or know a paper considering this topic?
Thank you in advance.
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Dear Jens,
if I have understood correctly, your objective is to find lymphocytes with speficic TCR that are expaded in tumor bearing C57Bl/6J mice.
I suggest you 2 paper in which they highlighted many differences in TCR repertoire between Balb/c and tumor bearing Balb-neuT mice after immunization.
Hope this help.
Best
Sergio
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Hello. Need help with (eBioscience) FoxP3 kit. I heard the external antibodies (CD4 and CD25) can be added, AFTER the fix/perm, after overnight incubation at 4C, CONCURRENTLY with the Foxp3 Ab.
Anybody tried it?
What would be my best controls? Single stains for each? CD4+CD25 either with or without Foxp3 (All-But-One control)? Isotype controls?
What percentage to expect, in human PBMCs ?
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There are many resources, where you can inform quite well regarding different surface stains using different protocols and different clones (e.g. CD4); I personally like cytobank a lot:
Or ebioscience to have a quick look...
Or BD...
Good luck and cheers
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Can you recommend a good LPS brand etc that you know works well or has been well established in publications? Can you tell me what medias I need to use to culture the splenocytes and IP cells. I have RPMI 1640 and DMEM currently available in the lab. What do I supplement them with? I've seen so many variations of the media in literature that I am confused. Also, if measuring cytokines after stimulation, best to measure secreted cytokines in media, or block secretion and measure cytokines in cell lysate?
Will be harvesting after stimulation and run through flow to characterize. Will also harvest some to use for mouse cytokine array.
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I agree with Isabella. You might also try Sigma for the LPS. As for measuring cytokine production using the supernatants for quantitating cytokine production by ELISA, multiplex, or other is good. Using intracellular staining/flow cytometry to look at single cell production is not quantitative for cytokine produced, but provides the frequency of cytokine producing cells. They are different data points and both may be useful depending on the context of your experiments or model.
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The cellsignaling one always give some non-sepecific bands and sometimes it even gives two close bands between 50 and 75KDa. I also tried with Novus and it also has two bands problem. Any one can recommend some others with the reference?
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You can try GP-62C from Progen.
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I am planning to study NFAT signaling in T cell and to isolate protein for the same. What is an optimum time for incubating a T cell with dynabead (I have read it can vary from 48-72h depending on exp). Also I read somewhere that the cell needs to be lysed before separating it from beads? How do I do that?
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By removing the beads magnetically you mean just to put them again on the magnet for 1 minute? Then pipet the cells into any other eppi? So following your suggestions  I don't loose activated T cells as the beads anyway do not stick any longer to the target (e.g. after 24hrs). Thanks in advance! 
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I'm looking for a good anti HNF4A for Chip qPCR and ChIPseq in mice, 
I'm working on MIM6 cells line and Langerhans islet, and the amount of HNF4A is low, but I don't want to give up that's why I'm looking for a very specific antibody against HNF4 for ChIP ?
Thanks, 
Tom
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active motif antibody for ChIP is very good.But  I am not sure if they have this product, you can go to their website and check.
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I am primarily looking for breast or lung models.
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Can't think of an exact place off hand.  Might want to check ATCC. 
Read through papers in your field.  Many researchers may have cells that would work for your purpose.    Additionally, many will just give you the cells so long as they are acknowledged and your lab just pays shipping.  It can be much cheaper.    And ask people around you at your institution.
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p21KO mice: does anyone have access to them and would like to collaborate?
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Thanks Rainer! Yes, i found someone in Spain willing to collaborate on this, so all good. But thanks anyway for your kind reply!
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I will be using ELISA for detection of protein. 
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CD34 for breast cancer
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Hi everyone,
I found that mast cell protease 1 and mast cell protease 2 expression level increased 4 fold (qPCR) in colon epithelial cells of KO mice, so here I want to ask, are there any potential signal pathway that can activate the mast cell and secret the protease1/2 in colon epithelial cell?  how to related this gene to mast cell activation signal pathway?
Thanks!
Xin
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Also
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.
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This thread is old, but I have a question to Bedrich.
Step 4 says dilute sample to 20ml with DMEM (1%BSA), is this when the tumor is small and digested in an enzymatic mix (serum-free DMEM plus collagenase) of less than 20ml? If that is the case, how about if the tumor is large (flank tumor ~1000mg) and the enzyme mix volume is already 20ml at 3.
Another point is regarding the specifications serum-free DMEM at 1 and DMEM/BSA at 4, what would the consequence be if one type (either serum -free DMEM or DMEM/BSA) is used through out? 
Still one more point (one before the last), I do not see DNAse in your protocol which I usually use to digest DNA released from dead cells and reduce sample stickiness; any explanation?
LAST POINT: I know of collagenase I and IV and that collagenase I is good when downstream analysis involves lymphocytes while IV is preferred when downstream analysis is on myeloid cells, but here you mentioned collaginase A and I would appreciate it if you elaborate on its use in terms of the points mentioned for collagenase I and IV.
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Hi everyone, my labmate and I have been trying to clone a few shRNA constructs in the past 2 months. We encountered quite a few issues and only successfully cloned 2 out of 18 constructs so far. I would like to ask for some advice from you. Thank you so much in advanced! Below are my questions and link to brief description of our protocol + result. Please let me know if there is anything else you'd like to know. 
Best,
Huong
- Our high background indicated that the CIP didn't work very reliably and that the vectors were not fully digested by both enzymes. How to fix this? Maybe to use other phosphatase? serially digest the vectors? 
- Do you have recommendations on how to make sure CIP works properly? If not CIP, how to efficiently get rid of/reduce the background?
- What is your protocol for annealing the oligos (temperature setting, buffer, concentration to start with, etc)? 
- How effective is the phosphorylation with T4 PNK? How to tell if the reactions actually work? Maybe by running gel?
- Did you follow NEB recommendation for total DNA concentration during ligation? If not, what did you usually use? And what is the optimal ratio of vector:insert?
- Out of the 9 positive clones that we got, 3 of them have some sort of mutations. Could the bacteria be accounted for this? If so, which other competent cells are better?
- Our successful constructs are for the same domains on two proteins. Could there be a favor toward these oligos? How to enhance the success rate of the others?
Link to protocol and result is attached! Thank you all!
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I am glad to hear that :)  
Chung Sub 
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Has anyone performed the optimal compensation of FITC-conjugated annexin V and Propidium iodide in apoptosis studies with flow cytometry? 
I think my dot plot is either under or over compensated.
Can anyone give me some tips on how to compensate this to reach acceptable visions of my populations?
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The standard curves for all of the trials for this experiment have had R^2 values of above 0.9. I need to determine whether this is acceptable or whether I need to troubleshoot and improve my technique for more precise results. I have been using an R&D Systems PGE2 ELISA kit.
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Hi Laura
I have attached the typical PGE2 standard curve given by R&D Systems on their website (assuming this is the same kit you are using). On a log scale the working range is still slightly sigmoidal in shape. I assume you are fitting a polynomial curve of some description to give the best fit to your calibration standards? If you are fitting a linear regression line to these standards it would explain a low R^2 value.
However, even if you are correctly fitting a polynomial curve to your standards, I would still be happy with R^2 being consistently above 0.9. You have proven by repeated trial runs that the kit is reasonably reproducible with respect to R^2 - this consistency is more important than the accuracy of your fit. While we have not used PGE2 kits, in our experience R^2>0.9 for an immunoassay is perfectly acceptable. It is more important to assess the accuracy of the kit in quantifying concentrations in your control samples.
Since this is a kit with pre-prepared reagents, there is little troubleshooting you can do other than proper storage, ensuring your plate washing is consistent and thorough and, as mentioned by Charles, that your pipetting technique is as good as possible.
Best of luck.
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Is there a scoring system available or developed by any member here for the interpretation of CXCL14 by Immunohistochemistry? Kindly share your views and insights. I am using this antibody for the first time. 
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I extend my gratitude Dr. Sihem for taking time to look for relevant articles, answering my question and for sharing really insightful articles for my help.
I failed to fetch any scoring system after going through few of the articles and hence was perplexed which way to design my small study of cxcl14 expression in different tumors. I wish to express more..ours is a limited facility department with no molecular facility. However it is there in other departments which can be collaborated with if need ed. At the most, we can do IHC here. I have purchased CXCL14 antibody and have just started applying on some paraffin sections.
Can you please elaborate on cohort of stained tissues please.
At last I thank you again.
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Immunohistochemistry
Immunocytocheistry
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Besides using the above stated reagents to permeabilize cells' membrane, you could use an another well known reagent ,saponin.
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I have used agapppe diagnostic kit for the estimation of serum LDH. In the procedure, 1ml of reaction mixture (4 ml of R1 + 1ml of R2) mixed with 10 micro litre of serum and incubated for 1 minutes at 37 degree celsius. But i didn't get the results. If any body using the kit for estimating LDH, could you please tell the exact procedure to be done.
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Miss Nicole, The principal of this reaction is based on NAD/NADH2 and 340 is the wavelength. I hope i may not get visible color reaction.
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I would like to address whether my genetically engineered human AD293 cells are more or less attacked by T-cells. Is there a human T-cell line that can be used for a cytolysis assay?
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Why not use T cell isolated from volunteers.  Just like mixed lymphocyte reaction. The cells line are convenient but may not show the truth. Good luck.
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What concentration and duration . 
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Even if the status of p53 is known in the cell line, one may require to optimize the concentration of hydrogen peroxide for a given time. Additionally, culture conditions (medium and growth factors) may influence the outcome. 
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I want to know the protocols to deplete MDSCs in mice bearing 4T1 tummors with particular emphasis on :
1. Which regimen of MDSC depletion (anti-Gr1 or Gemcitabine??) is better?
2. Dose and frequency of the depleting agent.
3. How long after the 4T1 inoculation is the first dose of depleting agent adminstered?
4. Is it possible to have suppressed MDSC expansion at all times during 4T1 growth or does MDSC cell population rebound at later stages of 4T1 growth? 
5. Is it possible to have selective effect only on the depletion of MDSC population without any effect whatsoever in kinectis of tumor growth?
Thank you.
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We use cimetidine 400 mg 2 x/day to reverse the MDSC. With patients.
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There are some software to predict a peptide's immunogenicity once I input my peptide sequence.For example, http://www.imtech.res.in/raghava/propred/index.html gives me a score of the peptide, what does the score mean? another software is http://www.cbs.dtu.dk/services/NetMHCIIpan/ ,which gives me % rank ,but what does it mean ?
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Both ProPred and netMHCPan are algorithms that predict the likelihood that a peptide will be bound by a MHC molecule.  This is important because peptides that are not bound by MHC will not be presented to T cells and thus cannot be targeted in T cell mediated immune responses.  It is also important to note that a peptide can be bound by MHC and presented to T cells and still not be immunogenic if the organism does not have any T cells bearing T cell receptors that recognize that peptide.
I am not familiar with the ProPred output, however I find the most relevant number to review in the netMHCPan output is the predicted "Score Aff(nM)" value (i.e. the predicted binding affinity for MHC).  In the tumor neoantigen literature, the standard convention (for now) seems to be that peptides with affinities < 500nM are considered MHC binders.  
In one study from our group, most peptides that were predicted to bind at 50nM or less were confirmed by binding affinity measurements, whereas about half of peptides predicted at 50-500nM were confirmed.
Hope this helps.
Benjamin
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Which depression model for rats do you prefer? What is the best way to induce depression in rats? We would like to start some projects in this field and we do not have any experience....
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You are most welcome, please let me know how it goes, and i might be able to help you with one of the Books (2010).   Best wishes.
Ghanim.
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Hello, 
I'm looking for an antibody against HNF4A that work for pancreas (that label HNF4A in the beta cells), I tried one from sigma and 3 from abcam, it works well in the liver but no signal in the pancreas.
If somebody can give me a antibody reference and/or some tips to make it work.
Thanks, 
Tom
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tanks for your tips, just few question, you permeabilized with port K before the fixation with PFA 4 % or after ?
And what il the concentration of the proteinase K ?
Thanks again
Tom
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I`m performing a flow cytometric analysis to study apoptosis in peripheral blood mononuclear cells (PBMCs) of patients using Annexin V-FITC (FL1) and Propidium Iodide (FL2). 
My tubes include and unstain, a control (containing PI and Annexin but left untreated), and several treatment groups. 
After I set the voltages and do the adjustment using the unstain and the control, the cells are displayed out of the plot while evaluating the treatment groups and I need to elevate the voltages of both FL1 and FL2 to bring the cells within the plot area. That`s while, when I set the voltages according to the treatment groups, my unstain and control are displayed in the positive area of both FL1 and FL2 in a sword-like manner! 
What is wrong? Is there a problem with my compensation?
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I presume this is because you suggest that the majority of the cells you isolate are leukaemic? It would be still useful to use a pan-marker to delineate a leukaemic population.
My group works on CLL, and the level of heterogeneity of spontaneous apoptosis is high. I would worry that all cells are PI+ without going through the double-positive stage first, particularly after 24h.
It could well be that you observe a toxic effect, rather than apoptosis.
Why not to stain for Ann+PI+  the cells straight ex vivo, then after shorter periods of time post-treatment with your drug, such as 1h, 2h, 4h. You should see no apoptosis as early as 1-2h post-treatment, so if the cells are dead, it would indicate toxicity. Your untreated controls should be the same as ex vivo. Likewise, if you use leukaemic marker, and your drug is specific for leukaemic cells you should be able to see if all PBMCs are dead (toxicity), or the leukaemic cells exclusively.
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I've cultured Treg cells in RPMI+10%FBS+IL-2,anti CD3/CD28 for 5 days,after this duratin I checked the cells' viability,but it was very low. my question is regarding the exosomes are released from cells to supernatant, is it made any problem in my work ?
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Hello Maryam,
Yes cell death definitely affects the exosomes...its like, entire profile change after cell death and there is also release of certain toxic cytokines into systems,..also if cells will be dead, then no healthy exosomes will be secreted as normal cellular processes will come to a halt...so, care is always taken that once you isolate exosomes, you always characterize the residual cells for cell viability and health status by Acridine orange staining or Annexin-pI apoptosis assay.That is must to confirm the viability of cells after exosome harvesting..So first, make sure that all your cells are healthy and viable and then only proceed with any of exosome related experiments...
Hope it helps !
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I am trying to detect IFNg-producing lymphocytes in human peripheral blood samples. I am using a viral stimulus which I guess is presented by the APCs present in the sample. Does anyone know if I should consider the amount of APCs and lymphocytes present in the sample to understand the assay results? If yes, which APCs should I quantify?
Thanks in advance
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... ni idea...
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Dear Colleagues,
Maybe someone could suggest mesenchymal stromal cells in vitro potency assays protocols to evaluate MSCs immunosuppressive activity. We are working with human bone marrow derived mesenchymal stromal cells.
Thank You
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Thank You for the suggestions
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Hi, everyone.
When I do flowcytometry analysis and calculate mean fluorescence intensity (MFI), I sometimes find the values are "negative".
Does that mean the staining was inappropriate or the intensity was nearly zero??
It would be fine if someone could tell me what does it mean.
Thank you in advance.
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I direct you to this interesting article from the Daily Dongle, a blog about flow cytometry from the same team that works on FlowJo flow cytometry analysis software.
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I am using the MabTechnology anti CD3/CD28 purified Ab for T cell stimulation, regarding the kit instruction I should add 0.1ug/ml of each, but I did not see any significant colony in my culture media.... does any one know about the optimum concentration of CD3/CD28 that is used for T cell stimulation?
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The following protocol we use for human cells: coat plate with aCD3 OKT3 (1 µg/ml in PBS, although 0,1/0,01 also works) incubate 4 hours at roomtemperature. Wash three times with PBS and add the cells to your plate.If we do a costimulation with CD28 (15E8, 5 µg/ml) in a pre incubation so cells in a small volume (to save antibody) and then adjust cellconcentration to the wanted concentration. Depending on what you want to analyze, activation molecules/cytokines you incubate for 4-72 hrs for cell proliferatiopn >4 days. After 3-4 days you should start to see clumbs. If it's pure T-cells you use, coating with aCD3 is essential, but also when you use PBMC coating is more optimal than soluble. Good luck
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As I was preparing the attached paper, it's mentioned that CD20 receptor is expressed in both normal and lymphoid cancer cells (page # 3, highlighted), yet, they designed monoclonal antibodies that target it...
How it can differentiate if it binds to both?
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I agree with Ellen. I'd like to add another point concerning B cell depletion therapies. Some chronic lymphocytic leukemias have relatively low levels of CD20 but express high levels of CD37. Thus, targeting a non-CD20 surface receptor may make sense, and not only that, the combined binding of CD20 and CD37 (which signals differently than CD20) may lead to more cell death as well as ADCC.
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I am looking for a murine intestinal markers. I have used Epcam1 but it seems to be affected by my gene of interest so I am wondering if there is an alternative markers to use for intestinal epithelial cells other than Epcam1?
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Thank you Stefan Koch. I wanted to use it for RT-PCR. I also wanted to check by flow if possible so thank you for your suggestion.
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Among the unfortunately (too) large set of chemoresistance mechanisms in cancer cells is the so "popular" ABC-related pump / MDR phenotype.
While there are hundreds (if not housands) of articles "explaining" how cancer cell poulations are enriched by one or another type of efflux pumps, how efflux pumps "eject" a cytotoxic compound out of the cancer cells, etc..., etc...
...
I request the help of the RG community to provide me with top-ranked articles explaining in a crystal-clear manner the biochemical / molecular pathways that lead cancer cells to activate the ABC system.
Are these molecular / biochemical pathways similar / identical to those leading to the activation of the ABC system in endothelial cells from the BBB, or in the intestinal epithelia of ruminants?
Thank you very much for your attention and your help,
Robert
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Dear Goodwin,
Your article (the one you attached in your previous response) is actually gorgious!!
I learned a lot and I encounrage RG members interested by "my question" to have an in-depth reading of your article!
Take care!
Robert
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it's said that everyday our bodies may have a large number of abnormal cells that may turn into a tumor of cancerous cells but luckily our immune system eliminate them before it's too late 
after a google search, I found out that the natural killer cells are the immune cells that are responsible for the recognition and elimination of abnormal cells now the question is how it's done exactly 
thank you in advance
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NK cells originally described as large granular lymphocytes, exhibited natural cytotoxicity against certain tumor cells in the absence of preimmunization or stimulation.
CD56 dim NK cells, which make up the majority of circulating cells, are the most potent cytotoxic NK cells against tumor cells.
  • NK cells directly kill target tumor cells through several mechanisms:
(i) by releasing cytoplasmic granules containing perforin and granzymes that leads to tumor-cell apoptosis by caspase-dependent and -independent pathways
(ii) by death receptor-mediated apoptosis.   Some NK cells express tumor-necrosis factor (TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL)
(iii) by secreting various effector molecules, such as IFN-c, that exert antitumor functions.
(iv) through antibodydependent cellular cytotoxicity (ADCC) by expressing CD16 to
destroy tumor cells.
  • Indirect NK-mediated antitumor immunity                                                       NK cells act as regulatory cells when reciprocally interact with DCs, macrophages, T cells and endothelial cells by producing various cytokines (IFN-c, TNF-a and IL-10), as well as chemokines and growth factors.
For More Details:
Cheng, Min, et al. "NK cell-based immunotherapy for malignant diseases." Cellular & molecular immunology 10.3 (2013): 230-252.‏
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i would like to investigate the movement of neutrophil in 96 well plate and i need to coat the well.
does anyone have experience about the best coat for neutrophil movement? BSA? HSA? FBS? PDL?
Thanks
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IF YOU COAT THE WELLS BY AGAROSE GEL YOU CAN SEE NEUTROPHİL MOVEMENTS UNDER AGAROSE.  THE END OF STUDY YOU WİLL GET OUT AGAROSE GEL AND STAIN WELLS TO SEE MOVEMENT TRACES ON THE WELLS.
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Does anybody know what are all the known receptors that can recognize and bind MHC molecules? Ofcourse there is the T-cell receptor and receptors on NK cells such as different KIRs...
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Thank you for both answers. Yes I am familiar with ILTs. However,  at the moment I am wondering whether HLA disparity could be theoretically recognized between allogeneic antigen-presenting cells (macrophages, DCs) in the absence of lymphocytes. This has not been addressed anywhere and in my opinion most likely does not exist in nature. But also true... nobody has addressed this so far...
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We want to obtain infiltrate lymphocytes in skin after Imiquimod (Aldara) treatment for FACs analysis. Some works used Liberase Blendzyme plus DNase I (Roche) and others used Dispase + trypsin/EDTA + Collagenase type 3. Has anyone used any of those? I there any way to obtain skin infiltrates? Does anyone have a  more detailed protocol?
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We use 10ml Liberase TL (307ul of 5mg/ml in HBBS) with 25ug/ml DNAse II. Our protocol is to add the liberase/DNAse mix, mince the skin into small (<2mm) pieces using scissors, add 25ug/ul dispase and incubate 37degC for 2 hours in a shaker.
Take it out every 30mins, then pipette up and down using a serological pipette to separate the clumps. After the 2 hours, add an equal amount of media (we use fibroblast), filter through a 70um strainer, pellet the cells at 400g 5mins, tip off supernatant.
This has given us the best yield vs. viability from any previous skin dissociation protocol.
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I am trying to use monosodium urate for immunofluorescence as a positive control in macrophages, however since they are insoluble crystals, I am trying to wash off the crystals not internalized but many still remain bound to the dish or non specifically on cells.
Any suggestions to overcome this issue would be helpful.
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Removal of unbound crystals from the surface of macrophages is quite straightforward with three washes with ice cold PBS supplemented with 5mM EDTA (remember that EDTA needs to be made up as a 0.5M stock solution first with warming and pH adjustment to ph 8.0 so it goes into solution). The washing protocol is described in our paper describing macrophage phagocytosis of MSU crystals in 2000 (Yagnik DR, Hillyer P, Marshall D, Smythe CD, Krausz T, Haskard DO, Landis RC. Arthritis Rheum. 2000 Aug;43(8):1779-89). Good luck with your experiments.
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Dear All,
I designed 5 panels of antigens to exam on flow-cytometry while each panel contains exactly the same fluorochromes. I noticed there are bleed-throughs in some of my channels even with compensation set up in advance.
The fluorochromes are as following: V450, FITC, APC, PE, eFluor 780.
The compensation is set up with both compensation beads and cells, both stained with single antibody. I choose either beads or cells for compensation setup based on brightness of the peaks. 
Since I still have bleed-though after compensation, I wonder if it is necessary to set up compensations for each individual panel. Does antigens have anything to do with this? Or is it just my compensation going horribly wrong?
Massive thanks in advance.
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When you use cells for compensation antigens may indeed have an influence. Are you using the brightest condition for each fluorescence during compensation? I mean if you are working with a marker that is increased upon stimulation, than your stimulated cell should be the sample used for compensation. But that shouldn't be a problem when you use beads.
I've never used eFluor780 but I read that it's excited by the red laser and detected by the 780/60 band filter. So I believe it might have some spill on APC (and vice versa). The same as for FITC/PE (but considering blue laser excitation).
What comes to mind is:
  1. Which instrument are you using?
  2. Does it come with suggested compensation values?
  3. Are your beads specific for this fluorochromes or are you trying to compensate different fluorochromes with similar spectrum (compensate FITC using Alexa488 for exemple)?
  4. Are you compensating manually? If you are, are you compensating visually or are you considering the median fluorescende intensity?
  5. Are you using FMO for gating control?
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I would like to know if there's a difference in cord blood between NVD and LSCS? I was advised not to use LSCS cord blood for ELISA and Western Blot. Is there any particular reason is to why it shouldn't be used?
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Dear Akshaya 
Please follw my collegue Ahmad Darwish , he is really expert in this area , his profile contains alot of his papers .
Good luck. 
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Is there any easy way to check  that?
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This is a major problem ocurring more and more often in labs.
Here attached you have two comments about this problem and also people who you could contact to bring you a precise response.
Best regards
Robert
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I am going to work with MCF-7 derived mammospheres. I need cholera toxin. Most companies sends in within 2-4 months. Any suggestion where I can get it? I am in Italy.
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thnx Baskaran
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Hi,
I am looking for syngenic tumor models for evaluating human-specific tumour targeting Ab in immune competent mice. More specifically, I am looking for models expressing either huHER2, huEGFR or huCD20 and which have already shown some therapeutic response to Trastuzumab, Cetuximab or Rituximab, respectively. I have identified potential models (TUBO-EGFR, BL3750), but they are not commercially available and I cannot get access to them. Do you have any suggestion? That would greatly help our research program!
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Dear Beatrice, Thanks a lot, that is very helpful!
Anne
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I am studying novel immuno therapeutic strategies in cancer and have had trouble in establishing a reliable system to study combination therapy with PD-1 blockade. I am wondering if anyone has had success with such a model. I have tried B16 tumors and EL4 tumors. Thank you.
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I realise this is an old thread - I'm having trouble getting hold of anti-mouse PD-1 (clone G4 ideally), would any one have access to this (and be willing to ship to Belfast, expenses paid!) or recommend a commercial alternative?
Thanks in advance!
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Hi dears
I am transferring in-vitro activated CD8+ T cells into host mice IV. 
They can enter the liver and accumulate around the infected hepatocytes expressing the antigen.
I am wondering which chemokines and chemokine receptors are involving in the homing of these cells in the liver and their accumulation around infected hepatocytes.