T Lymphocytes - Science topic

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!

can we genetically modify the memory b cells and t cells for a disease in order to make it work as vaccine.

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.

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.

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 :)

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

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.

We want to analyze whether T cells produce ROS under certain pahtophysiologic conditions.

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?

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?

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.

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

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?

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).

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.

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.

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.

What would be ideal mice strain to study T-cell dependent B-cell activation or KLH immunization?

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.

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

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.

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,

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

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?

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!

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?

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,

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?

hi

what are the most stable human CD4+ T cell line for transduction?

thank you

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!

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??

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?

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!

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?

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.

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?

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!

What are the normal ranges of B and T cells count from peripheral blood ?

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!

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

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

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.)

Can I use a normal CO2 incubator with some modification ?

I will really appreciate your help.

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!!

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!

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?

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 .

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!

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?

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

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!

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?

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

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?

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

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

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.

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! ;)

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.

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.

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!

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.

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

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)

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.

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!

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.

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

This is a methodology question for a new experiment I am trying.

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

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.

Is there a specific reason why splenocytes are used to isolate immunecells from?