Questions related to Cancer Immunology
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?
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.
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!
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.
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
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 :)
Master of Science in Biochemistry
K.J.Somaiya College of Science and Commerce
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 ).
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?
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.
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.
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.?
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.
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
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
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?
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.
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.
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
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?
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.
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?
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.
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
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?
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?
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?
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.
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 ?
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.
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?
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?
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 ?
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?
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.
- 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!
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?
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.
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.
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.
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?
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?
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 ?
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.
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?
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 ?
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
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.
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?
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?
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?
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,
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
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?
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?
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.
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.
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?
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!
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.
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.