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T Lymphocytes - Science topic

T Cells are primary agents in immunology and call mediated immunity. Helper, Cytotoxic, Memory, Regulatory, Natural Killer and gamma delta T Cells, their development, mechanism and application
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Hello everyone, I used several shScremble (from sigma, worldwide use) as a control for knocking
down my target genes in human primary T cells.
To my suprise, two shScremble I use has a negative effect on proliferation than just WT untransduced T cells and empty shRNA vector transduced T cells.
I am confused. Is there any bad things with shScremble? They should be the same with WT cell in theory.
I am wondering that what kind of control should I use? WT cells, empty shRNA vector or shScremble? Which one worth trust most?
Why people usually use shScremble rather than others?
Really hope someone could help to figure out.
Thank you so so so much in advance!
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This is because there are non-specific effects from shRNA. Using shScremble enable you to see specific effect of target gene knockdown.
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can we genetically modify the memory b cells and t cells for a disease in order to make it work as vaccine.
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Hi Pranav,
If there are memory cells available for a particular pathogen then it will do its work. To be clear memory B cells are generated during primary responses to T-dependent vaccines. They do not produce antibodies, i.e., do not protect, unless re-exposure to antigen drives their differentiation into antibody-producing plasma cells. As your question suggests the possibility of modifying memory B cells is not easy because the less percentage of that memory B cells would be difficult to isolate and maintain. If you are talking about modifying B cells there are many studies available for cancer immunotherapies. These B cells are used as APCs instead of DCs to avoid toxicity and generate an antigen-specific T cell response.
All the best
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I'm currently testing an Ab panel for T-cell analysis on Attune flow cytometer and I'm facing some strange stuff (see attached picture). I'm using IL4-BV711 Biolegend Ab for intracellular staining and when I apply this Ab I see a huge negative tail in the corresponding channel. I supposed some compensation issues, but it seems that it is not the case, since the tail appears when there is no compensation at all. Has anybody ever faced such kind of problem? What it might be? Too high gain on BV711-channel? I know, that flow data always contain negative values due to baseline correction after each event registration. However, in these data, the percentage of points that are below zero seems to be too high. Interestingly, the problem does not exist on the Fortessa cytometer. I would be grateful for any suggestions.
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This tail seams to be result of weak washing out of intracellular antibodies. I suppose cells from tail in SSC/IL-4 plot localizes on the right form "0" cloud. check with gates. How have You deterrmined the compensation, with splenocytes or beads?
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Hello,
I am trying to image live primary cells with Annexin V as a detector of PS exposure. I've currently been imaging in RPMI with 5% FBS, but I have realised that the calcium level of this may be too low for realiable Annexin V binding, as RPMI has a low calcium level of 0.42mM. Annexin V binding seems to require 1-3mM.
Other labs have successfully used complete DMEM with 10% FBS, would it be appropriate to use this for imaging even if I usually culture the cells in RMPI 5% FBS? Or should I try adding calcium (via calcium chloride) to my RPMI 5% media?
Thanks for any help.
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Hi Emily,
DMEM is more nutrient-rich and has higher levels of Calcium(around 1.6-1.8mM). DMEM generally comes in two variants, DMEM low glucose(1g/L) and high glucose(4 g/L). I would recommend you to culture your cells for at least 2 passages in DMEM High Glu and then stain them with Annexin V
Hope this helps!
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Hi everyone!
For human T cells isolated from PBMCs & frozen after isolation, how would you recommend to proceed with them after thawing? Shall I use them immediately (for sorting for instance) or let them recover (overnight or for a few hours) before experiment?
I need naive T cells, and I m not sure I can maintain this phenotype if I keep them too much in culture.
Thanks in advance for your help :)
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Once thawed, T cells must be rested for a minimum of 8 hours to a maximum of 18 hours to remove any apoptotic cells. A longer duration of resting could separate the dead cells and late apoptotic cells from the lymphocyte population. This would help to increase viability and improve functionality thereby enhancing the specificity and sensitivity of the assay (for instance, flow cytometry assay). The resting process could be helpful to restore the cells as well as have a positive effect on the recovery of the function of lymphocytes.
As you rightly mentioned, resting could also induce phenotypic changes.
Please refer to the research article attached below where in the investigators investigated the influence of resting on the phenotype and functionality of T cells comparing fresh PBMCs as gold standard to rested and non-rested cryopreserved PBMCs.
Hope this will be helpful!
Best.
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My Jurkats Cells looks differents after cytometry and even under the microscope. I just saw this and I am wondering what kind of contamination is this ?!
Thanks
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Thanks all for your answers ! Definitely Yeast ! I'll decontaminate all the lab and restart with a new culture !
Thx
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Hi all,
I am new to immunology field, need some help to distinguish CD3, CD4, CD8 and CD25. What are differences between them for T cells? How to decide which CDs are specific for T cells, B cells, neutrophils and macrophage.
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I commonly use CD3 to gate T cells; then CD4 and CD8 to gate T helper cells and T cytotoxic cells, respectively; Then using a CD3+CD4+ gating (Th cells) you could use CD25 and CD127 in combination to distinguish between effector T cells (CD127+CD25low) and regulatory T cells (CD127low CD25high).
You can check one of my articles for that: doi: 10.1038/s41598-019-43622-8
Best
Juanjo
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We want to analyze whether T cells produce ROS under certain pahtophysiologic conditions.
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Try this for method and alternatives
best of luck
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Hello everyone.
We isolated CD8+PD1+ T cells from the peripheral blood of a metastatic melanoma patient, with 99.5% isolation purity and 98% PD1 expression. We activated cells with Human TransACT "Miltenyi Biotec". After the activation phase had been finished, we left the cells in the expansion phase for 14 days, and then we performed a flow cytometry experiment to identify the phenotypic features of the expanded population. We found out that PD1 expression decreased from 97% to nearly 50%, but we still have CD8+PD1+ purity of 96%.
The part we didn't understand, what was the cause that led CD8+PD1+ T cells to decrease PD1 expression?
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PD-1 expression on naïve T cells is induced upon TCR activation. This transient expression decreases in absence of TCR signaling but is maintained upon chronic activation with a persisting epitope target. You used Human TransACT "Miltenyi Biotec", it may create a chronic activation on CD8+ Tcells. May be this activation results in lower expression of PD-1. Good luck
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Assessing differential expression of activation-related proteins in naive versus activated T cells is hampered by the fact that apples are compared with melons. Activated T cells vastly increase cell size, cytoskeleton, ER, protein synthesis and metabolism.
A problem occurs when you try to normalize to an internal control like actin, tubulin, Gapdh, etc. Despite equal loading according to Lowry determination all these proteins show increased expression in activated T cells a result understandable in light of the huge changes these cells undergo. What causes the bias? What would be a good loading control in this particular case? Histones, HDAC1? Can someone help with suggestions?
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Can someone recommend a good HKP for WB analysis
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I noticed that researchers use MOG35-55 to induce a type of T cell-dependent EAE, while for both T cell and B cell-dependent EAE, they choose rhMOG. Some papers report that B cells also contribute to the disease process in rmMOG-induced EAE setting. If these two recombinant proteins both activate B cells, I want to know what's the difference between them.
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Dear Echo Zhu
Please see the comparison below. There is a single amino acid substitution at position 42 of the MOG protein (position 7 of the peptide respectively) between the rodent (mouse/ rat) and human MOG protein from serine to proline.
rmMOG vs. rhMOG (aa 35-55)
rmMOG Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys
rhMOG Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys
The difference in B cell dependency of MOG induced EAE seems to majrily be attributed to immunization with full length (human) protein vs. the rodent peptide (in C57BL/6 mice), which may differ in other mouse strains (given their different MHCII) as per:
I hope that helps.
All the best and good luck with your experiments,
Michael
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i need clarify difference between ILC and T lymphocyte Th1,Th2 and Th17
because i found it have the same transcription factor?
like T bet, GATA3 and RORg
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If your ILCs are largely NK cells, there are a few ways I can think of to approach this issue. T cells express surface CD3, whereas NK cells do not; rather they express cytoplasmic CD3 only. T cells have TCR gene rearrangements, NK cells do not. NK cells express surface CD56 and the majority lack CD4 and CD8, though a normal subset will have CD8 expression. Give the close relationship between ILCs and T cells, I would expect a significant overlap in transcription factors, mRNA expression, et cetera. Please respond with more specific information regarding the ILCs, the tissue source of these cell lines, etc, if you would like to further discuss.
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I have activated human T cells in vitro (with CD3/CD28 for 1-2 days) and then stimulated them shortly with rhTNF (0-30 min). After CD3/CD28 activation, basal p-STAT3 levels incresead compared to the control T cells. However, upon TNF stimulation, preactivated T cells showed decreased p-STAT3 MFI. On the other hand, unactivated T cells had only slightly increased p-STAT3 levels. Any comments?
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Are your FACS data really convincing enough so that you can make those asumptions?
I would privilege Western Blot instead of intracellular FACS in order to make sure the signal that I look at really is coming from p-STAT3.
Also please consider the fact that activated T cells will endogenously produce TNF and that they produce IL-2 (which induces some STAT3 signaling), while unactivated T cells kept in culture for 2 days start/are dying (which could lead to a poor response to TNF).
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Hi everyone,
Been trying to culture murine T cells isolated from spleen or lymph nodes (iLNs) but been having difficulty in keeping the Pan T cells alive even after only 24hr of culturing (viability drops to ~20%). I process the tissue after harvesting with a plunger and 100um cell strainer in cold cRPMI to ensure single cell suspension, wash with PBS/2% FBS and performed RBC lysis with 3ml ACK lysis for 2 mins, quench with complete RPMI (cRPMI). Then isolate Pan T cells using the Myltenyi Pan T cell isolation kit (cat no:130-095-130 and stimulate with Myltenyi anti CD3 / or anti-CD3/CD28 beads or keep them resting for 48hr iin the 96-well plate at 0.25x10^6/well.
** Everything takes place at 4 C with cRPMI (10% FBS, 2mM Glutamine and 1mM sodium pyruvate, 100 units penicillin and 100μg/mL streptomycin and 1x NEA). For culturing i added to the cRPMI media: 0.05mM 2-Mercaptoethanol (2ME).
I have trouble-shouted the following and have NOT made a difference:
1) processing in cold cRPMI vs PBS or PBS/2%FBS (cRPMI elevated the viability by 20% vs PBS)
2) 2ME concentration: titrated between 50mM to 0.05mM and it seems that 0.05mM is the same with not adding 2ME (which was only around 20%) and anything above that concentration decreased the viability even more.
3) PBS tablets versus premade PBS from Thermo (pH checked)
4) stimulation induced cell death (although you always see some but even the resting cells die )
5) ACK lysis (optimised time required for lysis and quenching and volume) and also compared T-cells that went through RBC lysis (Splenocytes) vs cells not undergone through RBC (LN). Findings: had equally bad viability.
6) compared resuspending the cells with mechanical mixing (racking the tube with cells on a rack to release the pellet and then resuspending with the pippete) vs resuspending the pelleted cells with pippete directly . Findings: still did not make a difference
7) Centrifuge speed 1500rpm vs ~1200rpm (300xg). Did not make a difference.
Thank you all in advance, any help would greatly be appreciated.
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Hey Charys,
Maybe you can add some IL-2 to the medium? We recommend to add 50U/ml IL-2 in our T cell expansion protocol (see attachment).
Did you compare sorted T cells vs. seeding whole splenocytes? From my experience, the T cells are happier when you expand them in whole splenocytes (after a few days of expansion, you have around 90% T cells anyway).
I usually sorted CD4+ T cells and they didn't like it when the cell density was too low. So maybe you can also try to increase the cell density?
Best,
Alex
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Recently, we have done the macrophage antigen lpresenting experiment by co-culture of macrophages pre-loaded with OVA and CD4+/CD8+ T cells isolated from OT-II/OT-I mouse spleen. Using CFSE to stain the T cells, after 3-5 day co-culture, there is no significant differentiation of the target T cell.
Is there any probelm in the experiment? and is thre any trick in processing the sample?
Thank you very much.
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Hello,
I am looking to use Sytox Green as a substitute for propidium iodide (which I can't use with my microscope) in a live cell imaging experiment with T cells killing Daudi Lymphoma cells.
I know that propidium iodide has to be at high concentrations to enter through perforin pores (100uM).
Should I use the same concentration of Sytox green? Any help would be appreciated.
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SYTOX Green probe may be substituted for PI. SYTOX Green does not display any side effects on cellular viability, cellular proliferation or cell migration when used in concentration upto 1mM. You may use 150nM SYTOX Green. Please see the paper attached below.
Also, in another article attached below, the investigators explored the interaction of live cells with different fluorescent dyes, over extended periods of time in which cells were continuously cultured in the presence of selected probes: SYTOX Green (SYG, 1–1000 nM), SYTOX Red (SY-R, 1–1000 nM), YO-PRO 1 (1–1000 nM), PO-PRO 1 (1–1000 nM), propidium iodide (PI, 0.1-10 μg/mL), and 7-aminoactinomycin D (7-AAD, 0.1–10 μg/ mL) for up to 5 days. They found that treatment of several different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation.
Best.
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What would be ideal mice strain to study T-cell dependent B-cell activation or KLH immunization?
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You may use C57BL/6 mice.
Please refer to the two articles attached below.
Best.
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I am studying oxysterol effects on murine T cells and I want to perform a western blot on the gene that is more highly expressed. However, I am unsure which INSIG gene, INSIG1 or INSIG2, is more highly expressed in T cells. Please provide a citation if possible.
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Dear Natalie,
I hope you are doing well. Please take a look at this link (https://www.proteinatlas.org/ENSG00000186480-INSIG1/immune+cell).
Best regards,
Pooya
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I am investigating the role of the tumor microenvironment in shaping the antitumor immune response. In particular, we see that the T cell immune response is important but inhibited by some tumor-related factors. There is an issue that I was curious about but could not find the answer to: Some T cells remain resident in various tissues. Is this also true for tumor tissue? How long do tumor-infiltrating T cells remain in tumor tissue? Do they leave the tumor tissue and recirculate? How long do they come into contact with tumor antigens in tumor tissue? Does this contact time have an effect on the anti-tumor response?
In my research, I expose T cells to some of the factors they encounter in the tumor microenvironment, but I'm not sure how long I need to do them.
I would appreciate it if you could guide me on this.
Kind regards
Hasan
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Tumor microenvironement (TME) is very complex environment composed of various cells including tumor-infiltrating CD4/CD8+ T cells (TILs), and regulatory T (Treg) cells. The concern with TME is the mechanism of cancer cell evasion/escaping pathways via hijacking the immune system. Cancer interactions in the tumour microenvironment can stimulate biological components in the environment, such as tumour associated macrophages (TAM), cancer associated fibroblasts (CAF), and mesenchymal cells, which promote drug resistance against cancer. Some cancer biomarkers can also inhibit the production of tumour antigens, resulting in the failure of the targeted medication to attach/penetrate cancer due to cancer's microenvironment's unfavorable and complex mutational landscape, which also creates immunometabolic barriers.
In my point of view if we can block/inactive immune suppressor agents/immune checkpoint–related immune cells such as PD1/PD-L1, CTLA-4 would be a good strategy.
Many studies have reported that the tumor microenvironment plays a critical role in tumor progression and prognosis. Understanding the relationship between the various cellular components of the tumor microenvironment and prognosis is crucial for developing therapeutics aimed at the tumor microenvironment. Perhaps the following articles may be of use in gaining a better understanding:
Ji, X., Lu, Y., Tian, H., Meng, X., Wei, M., & Cho, W. C. (2019). Chemoresistance mechanisms of breast cancer and their countermeasures. Biomedicine & Pharmacotherapy, 114, 108800
Crespo, I., Götz, L., Liechti, R., Coukos, G., Doucey, M. A., & Xenarios, I. (2016). Identifying biological mechanisms for favorable cancer prognosis using non-hypothesis-driven iterative survival analysis. NPJ systems biology and applications, 2(1), 1-11.
Anfray, C., Ummarino, A., Andón, F. T., & Allavena, P. (2020). Current strategies to target tumor-associated-macrophages to improve anti-tumor immune responses. Cells, 9(1), 46.
Davis, R. J., Van Waes, C., & Allen, C. T. (2016). Overcoming barriers to effective immunotherapy: MDSCs, TAMs, and Tregs as mediators of the immunosuppressive microenvironment in head and neck cancer. Oral oncology, 58, 59-70.
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Dear all,
I am just trying to do an overall stimulation of PBMCs. Anti CD28/CD3 to stimulate T cells, but how can I stimulate the innate response? Some TLR agonist should be fine? TLR agonist can alter the natural T cell responses? Can I ask for some papers in which I can check this, please?
Also, if I am checking CD28 marker by flow cytometry, can the addition of anti-CD28 alter the detection of real levels of CD28 by flow cytometry?
Many thanks,
Julen.
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Hi Giacomo! Thank you so much! Hope everything is fine ;)
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Dear Experts,
We would like to inquire if planned to buy commercialized Anti-HER2/neu (369-377) T Cells to perform a cytotoxic T Lymphocyte (CTL) assay.
Do we need to use APC to activate T cells before CTL assay?
(Co-culture with antigen+APC first?)
We only have limited experience with CAR-NK cells.
In our experience, CAR-NK could use to kill the target cells.
However, we are not sure whether T cell needs to go through activation as we haven't found a proper protocol for the experiment.
Could you help us to solve the myth?
Sincerely,
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CAR-T cells are synthetic molecules in which the effector function of T lymphocytes combines with the ability of antibodies to identify specific antigens. Thus, CAR T cells do not require antigen presentation by antigen presenting cells (APC) and can recognize intact proteins. Therefore, the creation of genetically engineered T cells redirected to tumor antigens can bypass several mechanisms of immunological tolerance.
Please refer to the article attached for more information.
Best.
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Dear all,
We are starting a project that involves calcium imaging from activated T cells; in the literature people often use ratiometric dyes but I prefer to use OGB or calcium green AM
Does anyone has experience in loading theses cells with these dyes? can anyone reference a previously tested protocol? Many thanks
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I did use OGB BAPTA II already (octapotassium salt). However, I performed patch-clamp experiments (not sure if that's what you're looking for), so I loaded the cell by adding 50µM OGB in my internal solution. Worked like a charm!
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I have been trying to activate my Jurkat T cells using CD3/CD28 DynaBeads and looking at activation markers such as CD69 and CD62L. However, there have been no signs of activation after multiple experiments.
Do you have any recommendations to improve my results, preferably without adding PMA/ionomycin?
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Please consider the following suggestions:
Improve your cell culture medium by switching to "complete" medium, i.e. RPMI with glutamine, pyruvate, non-essential amino acids, beta-mercaptoethanol and Hi-clone FBS as per
which may improve your cell culture and activation results as per:
Please also consider the following paper.
As per Figure 5: CD62L is at best mildly upregulated, whereas CD69 does not seem to be upregulated at all following CD3/CD28 agonistic stimulation. They did culture for 24 hours in their experiment and used massspec as their readout, so therefore a time course might be a good experiment to do.
I hope that helps.
All the best & kind regards,
Michael
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I have been trying a protocol for T cell activation and differentiation using murine splenocytes in vitro, but I have been getting a lot of dead cells as indicated by flow cytometry. I am coating a 96-well round bottom cell culture plate with 5ug/mL CD3 plus 2ug/mL CD28 antibodies in PBS(--), at 100uL per well. The plate is then stored overnight at 4C. I had been making my CD3/CD28 solution as 5ug/mL CD3 plus 2ug/mL CD28 in 1mL PBS and transferring 100uL of this into each well, which would alter the final concentration that is actually coated onto the plate.
1) Is there an optimal range for each plate-bound CD3 and CD28 antibodies that should be added to the wells? I have not found consistent information for this in online protocols.
2) I have been using PBS without Mg/Ca, but one protocol uses PBS with Mg/Ca. Would this difference in PBS affect antibody binding to the plate?
I am wondering if the final concentrations of CD3/CD28 I have used are not enough for T cell stimulation and also wondering if these antibodies are actually plate-bound. Any help is much appreciated!
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Splenic T cells are stimulated with immobilized anti-CD3 and solubilized anti-CD28 antibodies in complete RPMI 1640 medium supplemented with 10%FBS, 50uM beta-mercaptoethanol, 1mM sodium pyruvate and 2mM L-glutamine in 96- well plate for 40-72 hours.
Most T cells will require 3 days to divide. These cells will clump but will not be attached to the plate. Harvest the cells using the cell scrapper.
The optimum concentration of anti-CD3 ranges from 1-3ug/ml and anti-CD28 ranges from 3-5ug/ml. You may add recombinant IL-2 (100ng) as it will stimulate the growth and differentiation of cytotoxic T cells.
Answer 1.
You should use anti-CD3 in the range of 1-3ug/ml. (1ug/ml anti-CD3 is preferable). Use solubilized anti-CD28 in the range of 3-5ug/ml, (5ug/ml anti-CD28 is preferable). You may coat the wells of 96-well plate only with anti-CD3 at 1ug/ml in sterile PBS. You could use anti-CD28 at 5ug/ml in the medium containing the cells.
After incubating the plate with anti-CD3 for 2 hours at 37 degree C or overnight at 4 degree C, you may aspirate the anti-CD3 solution. Do not wash the plate. Add the activated cell suspension in the anti-CD3 treated wells. Incubate the plate for 72 hours.
Answer 2.
Use PBS without Ca2+/Mg2+.
Hope this protocol helps!
Good Luck!
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It seems to be an apparent consensus that the activation and function of AhR influences the landscape of TC populations, but is there a group that is more severely affected?
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T helper 17 cells (Th17) are the most affected. AhR activation may directly or indirectly also modulate the commitment of Tregs, Th1 and Th2.
Please refer to the articles attached for more information.
Best.
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Has anyone assessed SLAMF7 (CD319) expression on T-cell ex vivo from human samples ? just wondering if a resting period after thawing cryopreserved cells can increase its expression and if yes, how long the rest should be ...
Thanks all,
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Please check the IMMGEN database for expression data on purified T cell populations from PBMCs. Unfortunately, it seems that there is very little SLAMF7 expression on T cells in general, at least for non-activated/ naive cells.
It seems only effector memory T cells show robust expression, but these you might have to re-stimulate, if you can't detect the ca. 20-30% in PBMCs in your healthy donors, as per:
Maybe that helps.
All the best & kind regards,
Michael
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Hello dear All! I had collected PBMCs from clinical patients and would like to seek for cytotoxicity activity of T cells with several intracellular markers by Flow Cytometry
I had set up unstimulated (plain cells from the clinical patient), and would like to include a stimulated condition with IL-18 and IL-12 but would like to know the right concentration for this, don't want to overstimulate them! Do you have any insights?
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You need to do a titration!
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hi
what are the most stable human CD4+ T cell line for transduction?
thank you
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You can use HuT78 CD4+ T cell line
Culture medium: IMDM + 20% FBS + 2mM L-glutamine.
Jurkat E6.1 T cells
Culture medium: RPMI + 10% FBS +2 mM L-glutamine
Please refer to the article attached.
Best.
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Hi everyone,
I am culturing Jurkat T cells E6.1 obtained from ATCC. This cell line is known to be in suspension forms. The cells were expanded as as suspensions for the first few days after reviving but then became adherent. The culture media used for culturing is made by ATCC protocols (RPMI + 10% FBS). Please let me know if anyone also experienced this and if there are any suggested treatment. Thank you for the help!
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One way to avoid adhesion of Jurkat cells is to culture them in flasks in upright position, which I always did. Nevertheless as they divide, they adhere to each other and form floating round aggregates.
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I am using lentivirus system to transduce my transgene in T cells. but I observed transduction efficiency goes down over the day in T cells. While The gene is stably expressing in HEK293.
Can anyone help please??
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Ankesh Jaiswal I am not talking about expression being truly lost. Expressing some genes can cause toxicity in cells. It could be possible that transduction isn't really going down in your cells but in fact the transduced cells are dying because expressing the gene (at this level) is toxic for them. So it appears like the transduction is going down.
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I have some PBMC samples and I want to activate the T cells to carry out some gene expression studies. However, my PBMC samples will not produce sufficient T cells if I isolate them as my samples are limited and I cannot collect more samples due to the pandemic. Are there any existing papers/protocols for activating T cells directly from PBMCs?
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I tried stimulating T cells in a pool PBMC by anti CD3/CD28 dynabeads. You can then confirm the expansion of T cells using FACS by staining CD3 marker.
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My goal is to establish an in-vitro culture of mouse splenocytes that are lacking in either CD4 T cells or CD8 T cells.
I tried doing this in the past by administering IP injections of anti-CD4 and anti-CD8 depleting antibodies into live mice on Day 1 and again on Day 2 before doing a harvest of splenocytes on Day 3. These splenocytes were then grown in-vitro for four days before analyzing them with flow cytometry on Day 7. However on Day 7 there still appeared to be a population of CD4+ or CD8+ cells showing up in the resulting FACS data.
My question is, did this arise because the depleting Abs were not working? Or could it be the case that CD4/CD8 T cells somehow have the capability to start re-growing again within the in-vitro culture between Day 3 and Day 7? If the latter is the case, are there any methods out there for depleting CD4/CD8 T cells starting with just an in-vitro culture of mouse splenocytes? Thanks!
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The method you have used can not deplete CD4 100%, there are some cells that will persist. The best to use knock out mice, if not try to use positive or negative selection kits for CD4+ cells before doing flowcytometry.
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We all use anti-PD1 or anti-CTLA4 to release the brake of immune response. And we all know that CD3/28 activator can always help T cell expansion and activation. So, why don't we use anti-CD3/28 as treatment in cancer setting?
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The main reason this is not done in vivo is that polyclonally activating ALL T cells via CD3/CD28 will cause CRS - cytokine release syndrome [also called cytokine storm] which is life-threatening and can be fatal.
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We're looking for the best system to test tumour targeted T cells against a cancer cell line. We've been using an Incucyte system that gives a fluorescent image based time course of tumour cell death. We're interested in what other systems, image based or otherwise, are currently available and reccomended.
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You can use Real-time potency assay for CAR T cell killing of adherent cancer cells.
Please refer to the link below for more details about the assay.
The eSight assay couples the simplicity, analytical sensitivity, and objectivity of real-time impedance monitoring with highly specific readout of live cell imaging to characterize CAR T cell-killing efficacy.
Hope this helps.
Best.
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Does PD-1 have to be expressed during the activation of T cells?
In other words, is it possible that T cells without the expression of PD-1 are already primed/activated?
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  • As we known, the naive B cell activation depend on T cell. The antigen alone cannot trigger B cell differentiation. However, whether the memory B cell could be activated by antigen without Th cell signaling?
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Actually, with certain antigens - ones that have a repetitive structure - primary B cells CAN be activated without T cell help. These antigens are known as T-independent antigens.
But memory B cells are absolutely dependent on T cell help. I spent several years as a post-doc working with Norman Klinman at Scripps on this precise topic. Look for a review by Phyllis-Jean Linton and Norman Klinman; she published that back in the late 80's early '90s. I do not remember the name of the journal.
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Hello,
I am looking for a database where I could find a list of genes that are responsible for the activation and recruitment of immune cells, especially T cells.
Most of the information I find is about genes that are specific to and expressed by immune cells.
Thank you in advance for your help!
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Dear Klaudia,
It is hard to answer your question without more context.
The first idea will be to check CXCL-9 and CXCL-10 by Elisa (these are chemoattractant cytokines for T cells).
Then it depends on what is your source of activation. If they are antigen presenting cells, you can check costimulatory markers on their surface (CD80, CD86, MHCII etc) or cytokines production (IL-12-immunostimulating, IL-10-immunosuppressive, by Elisa)
I hope this helps
Regards
Francesco
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What are the normal ranges of B and T cells count from peripheral blood ?
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Dear Chen
I concur with Malcolm. Ideally you should establish a normal range for your population since the values vary between countries, racial groups and even ages. In our laboratory, we use the following normal ranges for adults
CD19 (B cells) (cells/ul) 30-450
CD3 count 670-2180
CD4 count 480-1670
CD8 count 120-830
Good luck
Siva
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For the past few months, I have been troubleshooting a CRISPR KO of human CD3e in human primary T cells. I got the KO to work one time using 1250 ng TrueCut Cas9 (Thermo) + 250 ng (or 7.5 pmol) hCD3e sgRNA (from IDT) and using electroporation parameter: 1600V/10ms/3 pulses on the Neon Transfection System (10 uL Neon Tip). I got ~75% KO of the TCR.
Since then, I have been trying to replicate those results with no luck. I have tried different T cell donors, different electroporation parameters, a new Neon kit (buffers and tips), performing the electroporation on different days after stimulation, TRCB sgRNA, increasing the Cas9+gRNA amounts...nothing is working. I have gotten this KO to work consistently in Jurkat cells with ~90% KO efficiency (same hCD3e sgRNA). Soon I will test with a new Neon Transfection machine, more T cell donors, and human sgRNAs with different target sequences.
Has anyone also experienced difficulty in performing CRISPR KO in human primary T cells, but figured out how to get it to work consistently? I would really appreciate any advice! Thanks!
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Karen Christie ,,, many thanks for raising such an informative discussion. Actually, I am planing to also use Neon system the coming days, and I noticed your Comment regarding how washing your cells with PBS had a negative impact on your CRISPR transfection. Would you please further clarify what do you mean ???
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I have worked with HEK cells before and have done freezing and thawing with no problems. I am currently working with HEK 293 T cells to make lentivirus and after thawing them, I can grow them with no trouble in T75 flasks to approximately 80% confluency. However, the problem right now is that after I split the cells in the T75 flasks into 10 cm dishes, I observe a lot of dead cells floating in the media and only a minimum number of cells attached to the surface. The cells that have attached are still clumped together and don't seem to be growing. I have been very gentle with my cells and don't think that it's my protocol that's causing the issue but I could be wrong. I would greatly appreciate any advice.
In addition, I use DMEM 4.5 g/L D Glucose with 10% heat inactivated FBS and 4 mM Glutamax and 0.05% Trypsin with EDTA
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293T are very easy to remove from the substrate, use very low amount of detache agent.
Does 100mm culture dish are from the same manufacturer? If not, perhaps, the cells do not "like" the surface. Try new one
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Hello,
I have T cell populations that I need to view on both a dot plot and a histogram. I try to set the histogram axis so that histograms are stretched properly. Howerver, when I change the setting on histogram, the dot plot setting also changes and cells are getting stuck on the edge. So I would like display the same population with different graph axis settings.
Any tips how to solve this?
Thank you for your help
Vit
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You can duplicate the fcs-file and the compensation-matrix. Then you apply different compensation-matrixes for the two fcs-files and change the scaling for each.
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Trying to complete a textbook chapter on T cells, I would very much appreciate a description of the specific function of Th2 cells.
Hundreds of papers, even in 2021, is repeating the decades old perception that Th2 cells are responsible for the defence against parasites and type I hypersensitivity. But IgE production is controlled by Tfh cells these days, right?
Your help is very much appreciated! (Please include references in your answer.)
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Trim Lajqi Thank you for your answer and referring me to the two excellent reviews.
I must admit that I still do not understand whether it is Tfh or Th2 that direct immunoglobulin class switching to the production of IgE, but I acknowledge that there are other tasks fulfilled by Th2 cells. Best regards!
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Can I use a normal CO2 incubator with some modification ?
I will really appreciate your help.
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You 're welcome Leonor Huerta
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I'm trying to understand how lupus (particularly SLE) develops due to immunodeficiencies. I've understood that it's a type 3 hypersensitivity (autoimmune disease), where immunocomplexes (of self antigenes and anti bodies) accumulate, and that the immune system fails to degrade these immunocomplexes. I've learned at uni that the antibodies have self affinity for intracellular components (such as DNA, proteins etc.), and that T cells have self affinity for the intracellular components, so they recognize self antigenes and trigger immune responses? And does this happen because T cells have not been presented for self intracellular molecules in the negative selection (and they're not presented for even healthy people), or is it because the T cells in SLE pasients are not presented with self intracellular molecules in negative selection (and this is standard, and they should be presented with self intracellular components). Can anyone explain the molecular and immunological explanation behind lupus? Thanks!!
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You've hit upon one of those questions yet to be answered. And it hasn't been answered for many autoimmune diseases really. Diseases such as multiple sclerosis, rheumatoid arthritis, and SLE are postulated to be due to T or B cells with reactivity to self antigens but what initiates this is not clear. T cells and B cells reactive to self antigens can be found in both healthy and autoimmune individuals but there are mechanisms that compose a "peripheral tolerance". This would include T regulatory cells and the absence of danger signals. So a deficiency in T regs or a viral infection that breaks through tolerance and may activate T cells are possible means of initiating the disease process. Both genetic and environmental factors are influential. There is an association with specific HLA alleles for most autoimmune diseases so that's at least one component. There's a lot out there in the scientific literature so dive in! Happy to discuss further.
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I am trying to set up an in vitro experiment where I co-culture peritoneal macrophages and T cells together in the presence of HMPV infection. After incubating the macrophages and T cells together, I harvest the cells and run flow cytometry looking for T cell activation via Ki67 and CD44 markers.
I have repeated this experiment three times, adjusting the conditions between the three replicates and each time I see that the uninfected macrophages and T cells are more activated than the infected groups (i.e expressing more KI67 and CD44).
I tried removing mCSF from the media after setting up the co-culture since I thought this might be activating the macrophages. I also tried various lengths of times for the co-culture ranging from 48hrs-4 days. I added IL-2 to the media to help promote T cell survival. In addition, I use a 96 well u-bottom plate and added the macrophages: T cells in a 1:10 ratio to ensure adequate T cell activation.
Please let me know if you have any suggestions for what I should try next. Thank you!
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Dear Olivia,
your protocol for peritoneal macrophage isolation sounds good to me. I am a little bit curious about the purity. Before magnetic cell sorting we only get 40% of macrophages from the peritoneum (gated to CD11b and F4/80).
The other cells are mainly B cells and a few neutrophils. So when you plate your whole lavage without seperating the peritoneal macrophages, you will get a co-culture of your T cells with a mix of cells but for sure not only with peritoneal macrophages.
B cells and Neutrophils might not only influence your macrophages, but also your T cells. If not with secreted cytokines or other molecules, just because they will die over night and release DAMPS. So basically you confront your T cells with a undefined mixture of cells and molecules released into the medium.
I think a pure culture of peritoneal macrophages will solve your problem. And then you can infect them and you will only have the PDL1 upregulation as factor that might influence your T cells.
I hope that helps you. For any more questions please feel free to ask any time :)
All the best and stay healthy,
Marc
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I am investigating T cell responses to peptide-vaccine constructs in mice. The general scheme is 10 days after immunization (peptide X-CFA) at the base of the tail, I harvest the inguinal lymph nodes and isolate T cells. I co-culture the isolated T cells with irradiated feeder cells from the spleen of an unimmunized mouse together with no peptide (unstim) or peptide (stim). I also use a positive control of PPD (antigen in CFA). The ratio of irradiated feeders:T cells I use is 100k:400K (per 200 ul in a well).
I read out proliferation via CFSE on day 3 where I stain only the isolated T cells (not the feeders) prior to setting up the culture. The problem is, I seem to get quite a large amount of proliferation in the unstimulated condition in which there is no antigen/peptide. Further, I don't seem to be getting a response to the peptide I immunize with anymore.
I optimized my protocol over the past few months and I was getting quite good results in terms of proliferation. But ever since I switched to irradiating my feeder cells as opposed to treating them chemically with mitomycin C, it seems my assay is not working.
Is it a problem with my co-culture i.e. are my feeder cells not presenting the peptide? Why am I seeing such significant proliferation in my unstimulated condition?
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Chenglong Wang Thanks for you thoughtful response.
Let me know whether or not your mice colonies are infected. As for the foxp3 cells, did you find a specific source citing this? You are saying that in the absence of stimulation, these cells may induce low levels of proliferation among all T cells in culture? Please elaborate.
My proliferated cells show dye dilution as well as increased FSC. Please see my data attached below. This experiment compared using irradiated vs mitomycin treated feeders. I assayed both the LN and spleen of CFA-immunized animal and did a recall response with PPD. I compared an old batch of PPD we had (which works very well) and a new batch we recently got, which does not seem to work...Anyways, the point is to show you what my unstimulated and stimulated cultures look like in terms of the dye dilution and FSC. I am using cell-trace far-red (APC). These graphs simply come from initial gating on T cells followed by doublet discrimination. Note that you can see that irradiation seems to minimize feeder cell proliferation as there are less unlabeled (APC) cells in my gate compared to the mitomycin treatment.
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Usually I culture mouse T cell with RPMI media and the mouse EC cell lines(C166---adherent cells) always culture with DMEM. I want to establish a in-vitro transendothelial migration model that mouse T cells migrate through C166 monolayer. So I want to know if DMEM medium affect mouse T cells activity and function. Could I use DMEM medium to culture mouse T cells in order to adapt the DMEM culture environment .
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Hi Rouchan,
I have some experience culturing primary mouse endothelial cells and Tregs. As far as I know DMEM did not affect Tregs function in my setting (normal conditions, CO2 5%, 37C, etc). The co-culture lasted no more than 24hs. Despite my aim was not to do and in vitro migration assay I assume will not impact your T cells culture as well.
If you don’t mind me to ask, which T cells are you culturing? T helper cells? (Th1/2/17?) If is the case, you may want to run ICS flow cytometry as a control for any of the cytokines that these populations express (for example: for Th1-IFNgamma; Th17-IL-17, etc). In this regard you will make sure your cells are indeed functional. You can run a surface staining as well to make sure you have the right phenotype as well.
For ICS you will find protocols online for sure, I will copy one link just as an example: https://www.biolegend.com/en-us/protocols/intracellular-flow-cytometry-staining-protocol
Hope this helps, best of luck!
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Hi all,
I am trying to do a cell sort and collect equal numbers of CD3+CD8+ and CD3+CD4+ t cells from naive C57BL/6 mouse spleen. Theoretically 70-80% of CD3+ cells should be CD4+ and 20-30% should be CD8+, but when sorting the other day, the yield of CD3+CD8+ was only ~3% (we need at least 1 million, so this is not nearly enough), with 85% being CD3+CD4+ and the remaining likely be dendritic and NK cells. Based on the sort data, it is unlikely there was an issue with the antibody itself, nor the concentration used (I've also used it recently with no issues). Any thoughts on why I am experiencing significant loss of these CD3+CD8+ cells in comparison to the CD3+CD4+ t cell population?
Thanks for any advice you can provide!
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If a drug that's known to inhibit STAT3 and cause mitochondrial uncoupling results in cytotoxicity that is unique to SU-DHL-1 cell line but not in other cancer cells, what may be the reason for this?
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There are differences between cancer cell lines with respect to key mutations that play a role in many different biological processes in the cell like impaired apoptosis, increased drug efflux which could cause differing drug sensitivity.
Generally, cell lines appear to respond to drugs in two broad patterns.
One group of drugs, primarily targeting machinery involved in the cell cycle, protein chaperoning or DNA repair which elicit similar patterns of response across all cell lines.
A second group of drugs, trigger responses that are specific to cell type. Different cell types respond to these drugs in qualitatively different ways. Such drugs largely target the signaling pathways that are disrupted by oncogenic mutations.
So, wherever possible, cell lines should be selected that most closely resemble the genomic alterations of the tumor subtype being studied. No individual cell line can be representative of all cancers derived from a single tissue.
I hope this helps.
Best.
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Hello
I just want to ask if you can also recommend research or review articles highlighting the unique receptors found on CD4+ T cells that are not found on other immune cells?
Thank you
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Hello
Web results
CD4+ T Cell Subsets - R&D Systems
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Hi all, I did an IF staining for FoxP3 and CD3. Apparently, I can't do double staining due to the antibody available both from the same host. I just wonder if FoxP3+ cells must be overlap with CD3+ t cells? I saw more Foxp3+ cells than CD3+ cells in this case and wonder if the Foxp3+ I saw was a false positive. Any idea? Thanks in advance!
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FoxP3 + should be CD4+CD25+ Tregs. of Couse,FoxP3 should overlap with CD3+ T cells. usually CD3 should be expressed on the cell surface, while FoxP3 should be expressed in the nuclear of cells. they should co-expression in one cells. if you see they are differently localized. it means it is a unspecific binding and it is not correct. I often see some published paper they are wrong! confocal will detect them more accurately!
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I am trying to use " CFSE Cell Proliferation assay"
My experiment design is like this.
1-cultureing CD4+T cells
2-Stimulating withAntiCD3/CD28 beads
3-Adding exosome
4-evaluating the proliferation of whole  T-cells.
5-evaluating the proliferation of Treg cells.
I have to optimize the number of beads and exosomes in this experiment. my goal population is Treg cells.
I don't know how can I detect exactly the proliferation of Treg cells.
Would you please help me with this issue? Or if you think this experiment is not working, would you please give me some suggestions for the experiment design?
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I briefly wanted to reach out concerning your question.
Please consider the following publication for all things Treg cell isolation and culture.
In brief, they did use Ki67 for assessment of Treg proliferation. For some considerations concerning improved Ki67 staining in T cells, please check my earlier answer to a similar question
For a tried and tested protocol for CFSE staining, you may use the attached protocol.
For murine cells, check the methods section of
Article CTLA-4 control over Foxp3+ regulatory T cell function
All the best & good luck with your experiment,
Michael
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I am interested in activating T cells, more specifically Jurkat cells and study IL2 gene expression in the mutant cell line vs WT. Other than CD3/CD28, what are the other cell surface molecules that can be activated to get IL2 upregulation? I am not looking for PMA/PHA or PMA/io based activation of T cells. I will really appreciate if there are any reference papers for the same. Thanks
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Thanks Loka Raghu Kumar Penke . I think ConA will work well for my intended application.
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when designing a chimeric antigen receptor for cart t cell therapy can't we use a target which is also exist in T cells but express excessively in cancerous cells?
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I presume that you've found an answer to this question given all the recent publications in CAR T. To answer your question in short, yes, it should be possible, but why would you risk fratricide? Do you have a target in mind?
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Hello,
I am currently optimizing a naive CD4+T-cell activation protocol based on plate-bound CD3 and CD28 (both 4 µg/ml) in a TC 96-well plate.
I use RPMI (2 mM L-glutamine) + 10% FBS + 1% Pen/Strep + 50 µM 2-Mercaptoethanol as my medium when plating my cells.
The cells are incubated for 24h at 37°C and 5% CO2.
My question is the following: I only get around 50-60% live cells in my activation experiments (checked with FVS780). How do I improve this percentage? I have read that using IL2 may help (Miltenyi's protocol) but all other protocols that I find never mention this.
Thank you in advance and kind regards
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Please consider using RPMI supplemented with glutamine, non-essential amino acids, pyruvate and beta-mercaptoethanol may improve your cell viability during your T cell culture.
You can find the detailed protocols/ culture conditions here:
To further improve your culture conditions further, please consider your choice of FBS (Hyclone vs.ordinary GIBCO FBS), as per:
All the best & good luck with your experiments,
Michael
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I know rat antibodies are not well defined, but I will appreciate suggestions on which markers to look for IN RATS in case of the following:
1. activated T-cells
2. central memory T-cells
3. antigen-experienced T-cells
4. Th1, Th2, Th17 T-cell activation markers
Cell membrane or intracellular cytokine marker suggestions will be appreciated.
Thank you
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Please consider the following publications. In the methods section you have detailed lists of antibody cocktails and the respective clones used for flow cytometry.
I hope that helps.
All the best & good luck with your experiments,
Michael
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I am identifying cell types on Loupe Cell Browser using various cell surface makers. Are there any specific markers unique to NK cells that differentiate them from T-cells? Right now, I have filtered out the "NK cells" based on the fact that they don't express classic T-cell markers such as CD3E, CD3D, CD3G, CD8A, CD8B, TRAC; but do express genes required for cytotoxic functions such as GNLY, NKG7, etc.
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KLRF1 is the best gene to use, particularly after you're comformtable with your annotations of T cell clusters.
This recent 10x paper from Vivier also has a useful gene signature that works well
This is it (forgive the formatting as I pasted from R)
CD160", "CD244", "CHST12", "CST7", "GNLY", "IL18RAP", "IL2RB", "KLRC1", "KLRC3", "KLRD1", "KLRF1", "PRF1"
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Hi, dear γδ T cell researchers!
I'm looking for how to isolate Vdelta1 and Vdelta2 cells from peripheral blood and bone marrow, without stimulation, just isolate these two subtypes separately and then cultivate them and evaluate cytokine production.
I thought of isolating the total #gdTcell population first (using Anti-panTCRγδ magnetic beads) and then try to separate these two subtypes (Vd1 and Vd2) for culture in vitro, but I don't know how to proceed. I need to evaluate these subtypes without them being stimulated/activated.
I appreciate it if anyone can help me! ;)
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You can use fluorochrome-conjugated mAbs. First dye the sample with anti-CD45, anti-CD3, anti-Vd1 and anti-Vd2. I propose to use the Miltenyi, and use a MACSQuant® Tyto® cytometer. Second, you run the sample by positively selecting the Vd1 cells and obtain the Vd1 T lymphocytes in the positive well with sufficient purity, then you recover the sample from the negative fraction and run it again in another cartridge and positively select the Vd2 T cells. in this way you obtain the two populations of interest in a viable and quite simple way.
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Recently, I want to verify the certain treatment enhancing on antigen presenting ability of macropohages.
I searched the literitures, using OT.II T lymphocytes and OVA antigen, OT.1 lymphocyte and OVA antigen are often chosen to verify the ability on CD4 and CD8 T lymphocyte differentiation and proliferation.
I want to konw whether there is any other method to do the experiment. Because we do not have OT.II or OT.I mice.
How about co-culture the Naive T cells and macrophages in the presence of certain antigen "X". If this method is acceptable, what antigen "X" could be?
ps: Infectious and malignant diseases like tuberculosis and lung cancer are our intersted field.
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A follow-up idea: one way to look at a larger portion of the T cell population and response would be to use a superantigen, such as staphylococcal enterotoxin B [SEB]. Superantigens bind class II MHC on antigen-presenting cells and to multiple Vbeta regions on the TCR. I cite SEB because it binds to I-Ab on C57Bl/6 mice. The problem here is that your readout may only be detectable in CD4 T cells and not CD8 T cells, but I do know that SEB on C57Bl/6 APC triggers a significant proliferation response by T cells.
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Hello, I'm a student. I have a question that Why is IFN-gamma increasing while THP-1cell binding is reduced?
from the previous research Two very distinct cytokine secretion patterns have been defined amoung murine CD4+ T cells. Type 1 helper (TH1), but not type 2 helper (TH2), cells produce interleukin (IL)-2, gamma-interferon (IFN-γ) and tumour necrosis factor-β, whereas TH2, but not TH1, cells express IL-4, IL-5, IL-6 and IL-10. The different cytokine patterns lead to different functions of the two types of T cell. From the anti-inflammatory research I've read. He said Fucoxanthin could inhibit inflammation by reducing the binding of ThP1. cells, but with more IFN-gamma.
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Dear Chattramat!
Because IFN-γ reduces macrophage /monocyte motility.
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Our lab usually cultures T cells in flat or round bottom 96-well plates. I don't think these plates are good for pelleting the cells and changing media. I was wondering if I can culture my T cells in V-bottom 96-well plates, so that changing media is easier. However, I don't know if this will change the T cells' access to CO2, their proliferation rate, or influence their phenotype in a way that influences my experiment.
My T cells are activated with CD3/CD28 coated Dynabeads and subjected to various manipulations over 7 days.
Thank you!
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Hello Yifei Hu
Yes, you are right. Upon culturing cells in V-bottom 96-well plate, the cells will naturally localize towards the middle and at the bottom of the plate. This will limit the possibility of mass transfer in respect to the cells access to nutrients and oxygen resulting in an increase in cell death with decreased cell activity. This in turn may influence your experiment. The discrete surface geometry of the
V-shaped well will not suit the purpose (namely to culture T cells) in comparison to the flat or round bottom wells. So, I recommend you to keep using the flat or round bottom 96-well plates.
I also agree with you that flat or round bottom 96-well plates are not good for pelleting the cells and changing media. But if you master the skill in pipetting, I am sure you will be able to work with these plates.
Good Luck.
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Hi everyone,
Can someone share their optimal concentrations of PMA/ionomycin for spleen T cells?
I plan to use PMA/ionomycin as a positive control for T cell activation (surface markers, cytokines...)
Much help appreciated.
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We used PMA 50 ng/ml (Calbiochem, Billerica, MA) and 500 ng/ml ionomycin (Calbiochem) for C57Bl6/J derived T cells as per
There might be some value to do a titration experiment as the genetic background of the mice you are using, the environment and the specific functional readout (certain cytokines, co-stimulatory molecules, proliferation) may all be somewhat affected by the concentration you are using.
All the best & good luck with your experiment,
Michael
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I am currently investigating the potential of using tumour lysate as a source of antigen in cancer vaccines.
I immunized mice with microparticles (MP) containing either EL4- oder EG-7-lysate and polyI:C. 6 days post-immunization I performed an IFNy ICS and ELISpot, where I restimulated with different peptides. In ELISpot, I often observed very high background in unstimulated splenocytes from lysate-immunized mice. Interestingly the same splenocytes did not show any substantial background in ICS.
Do you have any idea how this could be? The only difference between the two splenocytes was I depleted them of red blood cells before using them for ELISpot. Another thing could be incubation time (5h for ICS, 18h for ELISpot), but I don't see how that could activate T cells.
I would appreciate some input if you have any ideas.
Greetings,
Isabel
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I have some experiments using flow cytometry on db/db mice kidneys.
-During the steps of processing into single cell suspension, I use collagenase A (Cata#10103578001, Sigma) to digest the mice kidney: 1.5mg/ml Collagenase A in RP10 (final volume 2.5ml). Then I use percoll to yield lymphocytes in the kidney.
-During the staining steps, I use BV510 CD4 for staining of CD4+ T cells.
-For fixation step, I use 2%PFA, and let it sit for one week before I run it.
However, I always get the graph where i can't see the separation of CD4+ cells in the kidney.
Can anyone give some advice? (pic below showing the gating for CD4& CD8 for spleen and kidney)
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Hi Lingyun, It is very likely that you are losing the surface epitope recognized by your CD4 as a consequence of the digestion protocol. If you are doing intracellular flow for cytokines or transcription factors, you can add CD4 to the intracellular cocktail of Abs. Alternatively, you can rest the cells for a bit after digestion and before staining. Otherwise, you end up having to trust the CD3+CD8- fraction to be Th cell, but as CD8 is also somewhat vulnerable to some digestion protocols, that is less than ideal.
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Hi folks,
Currently, I made construct to generate version 3 CAR-T cell. Before IVT cell, I tested expression in 293t cell. It was good and highly expression.But not express in T-cell. Does anybody know what happened or any experience in this situation? Thank you all.
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from google:
T lymphocytes are considered as a hard to transfect primary cells. - T cells are resistant to common chemical transfection reagents, such as Lipofectamine 2000, polyfect and TurboFect. - These chemical transfection reagents are less popular in the case of T cell transfection due to their low transfection efficiency
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My two intependent variables are IFN-y and IL-2 T-cell responses to a given antigen. My dependent variables are 10 different antibody titers for that antigen in serum and saliva. I want to analyze the correlation between T-cell responses and antibody titers. Should I be performing a multivariate multiple regression analysis for this? How can I do this on Prism?
P.S. Most of my data is not normally distributed
Much thanks!
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Good day, Philip Samaan
Multivariate multiple regression is absent in Prism. You can do correlation analysis, Spearman (r), or may use logistic regression.
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Hi,
I am currently treating T cells with CD137-targeting aptamer to examine if the aptamers can specifically bind to CD137 receptor to be internalized.
I performed my experiment by treating my T cells (both wildtype and CD137 knockout T cells) directly with the aptamer for 24h. The cells were then lysed, followed by RNA isolation and RT-qPCR to detect the presence of the CD137-specific aptamer. However, CD137-knockout T cells which supposed to be negative) and wildtype T cells show positivity for the presence of aptamers.
I assume that this could be due to the unspecific binding of the aptamers to the cell surface. So I would like to ask for suggestions if there is any RNase product that I can use to remove cell-bound aptamers prior to RNA isolation.
Thank you.
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Steingrimur Stefansson Thank you so much. I will try with proteinase K first and check on other methods if proteinase K does not work well.
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Hello Everyone,
I am facing trouble to get maximum yield of T cells and NK cells from from female reproductive tract mucosal sampling especially from Cervicovaginal lavage (CVL), please share your experience and expertise in this regard.
Thanks in advance for your help.
Thanks,
Sakthi
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Dear,
I hope t article attached will be helpful for you.
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This is a methodology question for a new experiment I am trying.
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Hello,
You can do a stimulation overnight of your monocytes, then the next day you wash twice with pbs before adding new media with your T cells stained with CFSE (or another cell tracer to check the proliferation). Then check proliferation or other markers after 72h of co-culture.
Remember that activation of T cells is MHC dependent (unless you use pma/ionomicin), so to have good results you should use an antigen (like OVA, to pulse your monocytes with) and use OT-1 T cells to check for proliferation.
Without a specific antigen, your proliferation will be quite low.
Regards
Francesco
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I would like to determine whether my vaccine candidate is able to elicit a cellular immune response in Balb/c mice. I was thinking of isolating the splenocytes from the inoculated mice, stimulating them with the peptide antigen and then doing FACS to determine the numbers of CD4+ and CD8+ cells that have proliferated. How long should I incubate the antigen with the splenocytes before harvesting the T cells for FACS?
I would also like to do a cell-mediated cytotoxicity LDH study to determine whether a cytotoxic T cell response has been elicited. In this case i would like to incubate the macrophages from the isolated splenocytes with antigen and then add the T cells from the splenocytes the following day to determine whether they are able to kill the macrophages. I was thinking of just incubating the T cells O/N in culture medium before adding them to the peptide-stimulated macrophages the next day. My questions are the following: Should the T-cells be stimulated in any manner prior to the addition to stimulated macrophages? Also, how long should i incubate the T cells with the macrophages before measuring the LDH released?
I am looking forward to your responses,
Daria
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Thank you very much Martin. Your help and advice is very much appreciated. I will let you know how things fare:-)
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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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Is there a specific reason why splenocytes are used to isolate immunecells from?
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