T Lymphocytes - Science topic

Dear, In my experimental model I will administer a substance intraperitoneally and some time later I need to collect the T lymphocytes from the spleen for phenotypic characterization in order to evaluate the host's adaptive immune response. I need to know: i) on average, how long after administering the substance I should collect the spleen; ii) how to isolate T cells from spleen and, finally, which T lymphocyte activation surface markers I should use (in addition to CD8; CD4 and CD69).

We are aware that T cells can specialize into various memory cell types, such as stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and resident memory (Trm) subsets. However, the intracellular signaling regulation involved in the differentiation of these subsets appears to be less understood. Are there any evidence indicating important signaling pathways or key factors that might influence or determine the generation of specific memory cell subsets?

I am currently performing co-culture experiments with murine CD8 T cells isolated from mouse spleens and with skin cancer cells (PDV) in a 24-well plate. I first isolate the CD8 T cells from spleen which are around 70% alive at time of isolation, mix them together with CD3/CD28 T Cell activation beads to activate the CD8 T cells, and plate them 2x10^5 T cells on top of 2x10^5 skin cancer cells that were plated the night before. The cancer cells are treated with mitomycin C prior to plating the T cells in order to halt cancer cell proliferation. Then I let them incubate in a tissue culture incubator with standard parameters (37 degrees Celsius and 5% CO2) for 5 days. In addition to the wells where I co-culture the T cells with the plate cancer cells, I also plate some of the T cells with the activation beads in an empty well to act as a control. I then assess the viability of the T cells by staining with a live dead stain and running flow cytometry. However the results of the flow cytometry show show that most of my T cells are dead (<40% viability). The most problematic issue is that the T cell-only wells come up only 1% viability which render the rest of the experiment null since I would not have a T cell control to compare the T cell viability from the T cell/cancer cell co-culture wells.

The interesting observation is that the T cells co-cultured with cancer cells have greater viability compared to the control T cells cultured by themselves which makes me think that the cancer cells are stimulating the T cells and improving T cell viability that way. I was wondering if there are any adjustments I could make to improve the viability of my T cells especially for the T cells in the T cell only control wells?

Some adjustments I have made already:

1. Increase the speed of the T cell isolation from the spleen to improve baseline viability prior to seeding on the cancer cells

2. Utilize wide bore tips when pipetting T cells to decrease the shear force on the T cells

Thank you for any suggestions and apologies for the length of this question.

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 .

We are planning to check the level of phosphorylation of NF-KB in the CD4+ T cell population after activation with anti-CD3/CD28 and treatment for 15 minutes. However, I constantly see two fixed bands with MW around 65 and 25 KDa which might come from serum proteins but when we removed FBS from the media, they are still there. Since we're looking for p-NF-KB in 65 kDa size, how can I fix this issue?

I work in a project where we need to knockout a gene at primary T cells targeting an exon that is shared between all variants. However, while the sgRNA design has been optimised ,we still see low KO efficiency. (Note: we are using RNP method and electroporation)

The untransduced T cells are produced by mock lentiviral transduction of human primary CD4+CD8+ T cells. These cells are subjected to comparable manipulations as CAR-T cells: activation, spinoculation (without lentivirus), and expansion. These T cells are meant to be negative controls in experiments using lentivirus-transduced primary CAR-T cells.

Why not to use Scramble or a vector without a construct? Why UTDs are used?

I'm currently working with human primary T cells, isolated from whole blood that I get from my university's blood bank, and I'm trying to determine if my experimental treatment can enhance cytotoxicity. I'd like to run some type of in vitro killing assay but am struggling to find a good target cell for the cells that I work with.

In general, I use cells that are not CMV-positive. I've seen lots of literature using T cells from OVA mice and having the target cells pulsed with OVA peptide, but something like this isn't feasible with the cells that I have access to. Does anyone know if it's possible to run a killing assay of this type with run-of-the-mill human T cells? Is there a good target cell line that any human T cell with kill?

Thanks in advance!

Does anyone have a protocol for measuring T lymphocyte activation, either by flow cytometry or by measuring cytokine production? I'm working with CAR-T lymphocytes and I want to see if incubating them with patient sera will activate them in a specific manner. Can you suggest different cytokines and activation markers?

Hi- I would like to transiently deplete CD8 T-cells in vivo to allows allografts to establish, but am testing T-cell activation as a therapeutic modality, so need them to come back with appropriate timing. Does anyone have experience with re-population kinetics of T-cells after in vivo administration of mouse anti-CD8 mAb clone 53-6.7?

I have attempted various transfection reagents including Lipo3000/Lipo2000/Hieff Trans™ Liposomal Transfection Reagent, both GAPDH positive control and custom siRNA targeting gene of interest at different concentrations (20nM, 40nM, 60nM) in a 24-well plate, FAM-NC, and time points (24h and 48h post-transfection) for numerous trials. However, none of these approaches yielded the expected results. Could it be that non-electroporation methods are not effective for Jurkat T cells? Or does anyone have a more effective and unique experimental protocol? I'm hoping to receive your guidance. Thank you.

I am trying to expand TH17 cells from Naive T cells for the first time. Can you suggest the best expansion kit available in the market to do the same?

In mouse SI-lamina propria T lymphocytes stimulation with PMA (20ng/mL) /Iono(1ug/mL) is not strongly inducing IFN-g after 6hrs. The previous results in our lab had 4-5% and I could have just 2% max, though the protocol and antibodies to stain everything is followed in same way! any suggestions or comments would be greatly appreciated.

Bref A (1ug/mL) after 2hrs of PMA7Iono.

I want to deplete CD3 T cell from PBMC, how can I do that without magnetic bead separation?

Hello all,

I'm looking to measure Ag presentation to T cells on a B cell line HLA. I was hoping that I would be able to conjugate my Ag to a protein tag (FLAG, GFP, etc) and get a functional T cell read out as a proxy for presentation of my Ag. But as far as I can tell there isn't a T cell line with a TCR that is specific for anything like that.

Does anyone have any thoughts or know of any cell lines? I'm working with HIV so an Ag specific T cell line isn't really possible.

Thanks!

I've been trying to activate and expand naive T cells that have previously been isolated from PBMCs with a pan-T cell isolation kit from Miltenyi and cryopreserved in 90% FBS 10% DMSO. I have decent viability post thaw >85% with ~10% loss in viability from the pre-freeze viability. However, over the 10 days that i'm expanding the T cells, I initially see good activation, but then the T cells start dying off and I end up with abysmal viability by day 10.

Here's the protocol i'm using:

I rapidly thaw the vial of T cells in the water bath and when the vial is almost completely thawed, add 1mL T cell media dropwise. I transfer the full volume to a 15mL tube with more T cell media dropwise, then spin at 300xg for 5 minutes. I activate the T cells in Miltenyi TexMACS medium supplemented with 300 IU/mL IL2 and CD3/CD38 Dynabeads at a 1:1 T cell:bead ratio. I plate the cells at 2 million/mL in a 24 well plate for the initial activation. I expand the T cells for 10 days, checking the viability every 2 days and replenishing media/cytokine if the media starts looking yellow, keeping T cells between 1.5-2 million/mL.

This protocol has worked very well for me from T cells isolated from freshly prepared PBMCs, but hasn't been working well with the cryopreserved T cells. I initially see very good clumps forming that get larger, but towards the second half of the 10 days the clumps don't get very large and the T cells start dying. Over 10 days I had less than a 2-fold expansion, whereas with fresh T cells I could get a 4-5 fold expansion over 10 days.

Does anyone have experience activating and expanding thawed naive T cells or have thoughts on why my T cells may be dying? Whats weird is that I've used the exact same vial of frozen naive T cells to generate CART cells using a 3 day Dynabead activation with IL7 & IL15 followed by viral spinoculation, and the viability of these CART cells is fine after a few days of expansion after spinoculation.

Hi! Could someone help me to solve this problem?

I will coat the 6-well plate with anti-CD3 first. Then, add anti-CD28 into T cell culture and remove T cell culture into 6-well plate. However, Jurkat cells always form clumps. Do I need to split the T cells before adding anti-CD28 into individual cells? What should I do? Centrifuge the cell culture?

Besides, I will then test the CD69 and CD25 expressed on activated T cells by flow cytometry. In this test, I will add anti-CD69-FITC and anti-CD25-PE to the cell culture. Do I also need to centrifuge the T cells to split them from clumps?

Thank you in advance!

A reviewer wants me to generate CD4Cre CD19Cre cKO mice to complement the individual Cre data in an initial submission. I think that breeding and genotyping here could be a long and painful process. Does anyone know of a Cre driver that is 'pan'-lymphocyte?

Many Thanks in advance

Activation of naive T cells generally requires antigen to be presented by dendritic cells in lymph nodes. Activation of naive B cells generally requires opsonized antigen to be displayed by follicular dendritic cells in lymph nodes.

Thus, it seems that dendritic cells and complement (innate immune system) need to recognize a pathogen before an adaptive immune response can be initiated. Is this always the case?

The isolated T cells will be further activated and expanded in vitro for transduction (with chimeric antigen receptors). The ex vivo culturing will take about 2-3 weeks.

I know that for isolating rare cell populations (I.e. pDCs) it is recommended to pool spleens together to obtain sufficient numbers but not sure if the same can be done for mouse T cells. Would there be any concerns of cross-reactivity despite the cells coming from the same background (C57Bl6 in our case) only different individuals?

How many naïve T cells we can get from a C57BL/6J mouse spleen and lymph nodes? or in other word, what percentage of the cells from spleen and lymph nodes are naïve T cells?

I am doing multiplex immunofluorescence assay on FFPE section. I used CD3 to identify T cells and observed that some of T cells stained with CD3 showed nuclear staining instead of membranous. I am not sure why the staining pattern is nuclear for CD3? is it possible to see nuclear staining with CD3?

Why is tetramer only used to test the interaction between T cell and peptide-MHC complex and not to test binding affinity between peptide and MHC?

Hello, I isolate PMBCs from blood using Ficoll and plate the cells in a 96-well plate for different treatments for a few hours before T cell activation. At the end of the treatments I centrifuge the plate, discard media and use new media to pipette the cells several times and transfer to an anti CD3 coated 96 well plate for 3 days, new media is also supplemented with anti CD28 ab and IL2.

I can't seem to get the cells into the new plate and rarely get any cells in the plate after the 3 day incubation. I was under the impression that these cells detach easily with pipetting. Any suggestions?

First, we coated 96 well TC-treated plates with 5ug/ml anti-CD3 antibody (17A2, biolegend)

Then, we added Naive CD4+ CD25- CD45RB high T cells (500000) in each well after sorting through flow cytometry. Next, we used anti-CD28 antibody (soluble 2ug/ml, biolegend). Again, different cytokines such as 5ng/ml TGF-β, 20ng/ml IL-6, 10ug/ml anti-IL-4, 10ug/ml anti-IFN-gamma were added in each well. After 5 days, when we detecetd TH17 cells after, we found low ratio of IL17+ cells (1%-2%) and most of cells were dead (>80%). This is the main problem. Thats why we want to know which can cause this problem.

They don't take up any dyes, and they don't activate when treated with IL-2. I've confirmed it multiple times that these cells are CD4+ T cells by immunophenotyping (FACS). So why won't these cells take up any dyes? Or lenti? Why do they look like opaque black spots under IF?

Argh!

Hi,

Does anybody know whether effector or memory T cells can de-differentiate into naive T cells, rather than just become exhausted or undergo apoptosis? Thanks

Recently I’ve started seeing these clumps in my primary MSC flask (T175). I first noticed these compact clumps in a flask near 80% confluence but I’m also seeing it in less confluent flasks. The clumps vary in size. The medium is neither cloudy nor changing color. I’m using a basic growth media (DMEM, 10% FBS, and 1% antibiotic-antimycotic). When passaging I dissociate the cells using 0.25% trypsi-EDTA for 5 minutes. I’ve been washing the flasks with PBS + 3% antibiotic-antimycotic at media changes, but have not seen any change. Does this look like fungal contamination or could it be cellular contamination (for example, T cells or pluripotent cells clumping) or cellular debris? Whatever it is, it appears to sit a a level above the live, attached cells (with other dead cells).

Thank you!

I isolated CD3+ Cells from frozen PBMCs and then I seeded them in a 96-well plate in different conditions with some independent variables.

In this experiment, I need to incubate the plate for 7 days and then I do FACS analysis.

The problem is that after 1 week I lost around 70-80% of cells in each condition.

What should I do to lose a low number of cells during the incubation?

Is it possible to transduce a human CAR into mouse T cells and have the mouse T cells function when the CAR binds it's antigen? I am thinking that if anything the singling domain of the CAR, as it is human, may be a problem for the mouse T cells.

Hello,

I have been trying to do a lentiviral transduction of mouse primary CD4+ T cells with little luck. We do not see any expression and cells begin to die out.

Current protocol:

Isolate CD4+ T cells, transduce on day 7 with spinoculation (1000G, 1hr)

Thank you!

Hello all,

I could really use some advice with T cell intracellular cytokine staining. I've been trying to stimulate CD4+ T cells isolated from mouse spleens and stain intracellularly for cytokines, including IL-2, IFN-gamma, and IL-17A. However, I see very poor cytokine staining in my stimulated T cells, even IL-2 staining. I expected that most of my stimulated T cells would be positive for IL-2 intracellular staining, but only about 3 percent of my T cell population are positive for IL-2-PE when I run my samples on our flow cytometer. I see clear activation of my T cells not treated with Brefeldin A based on increased percentages of T cells positive for CD69 post stimulation, so I doubt that stimulation is the problem. If anyone has tips or tricks on optimizing T cell intracellular staining to share, then that would be most helpful!

Below is my general protocol:

1) Isolate CD4+ T cells from mouse spleen by mashing spleens and using StemCell EasySep CD4+ T cell Isolation Kit on the spleen lysate (I usually get 3-6 million T cells depending on the age and genotype of the mice)

2) Plate CD4+ T cells in 24-well TC-treated plate coated with anti-mouse CD3 antibody (5 ug/mL); soluble anti-mouse CD28 antibody is added (5 ug/mL); usually 400,000 T cells in 1 mL of R10 solution

3) Incubate T cells for 30 minutes at 37 degrees Celsius

4) Add 1 microliter of Brefeldin A (1,000x stock solution) to wells; I've also tried 0.5 microliters

5) Incubate T cells for 6-12 hrs at 37 degrees Celsius; wash after incubation with PBS/BSA

6) Stain cells for cell membrane markers (CD4, CD25, CD69) for 30 minutes

7) Incubate cells in BD Cytofix/Perm for 20 minutes; wash with BD Perm/Wash

8) Stain cells with intracellular cytokines (e.g. IL-2-PE) diluted in BD Perm/Wash for 30 minutes (1:50 antibody dilution)

9) Wash cells with BD Perm/Wash

10) Resuspend cells in 1xPBS 1%BSA

11) Ran cells on BD Accuri Flow Cytometer, collecting 50,000-100,000 events

12) Analyzed flow data and performed compensation using FlowJo software

I'm new to the cancer immunology field, and trying to develop an antigen dependent T cell : cancer cell killing assay in vitro. I plan to use OT-1/ova. I would isolate T cells from OT-1 transgenic mice. Do I need to use OT-1 homozygous or heterozygous T cells for the T cell killing assay? Thanks in advance!

T cells

Recently, I am working on a project that stimulate the TCR- transduced Jurkat with peptide, and looking for a activation marker (cytokine or surface marker). What is the activation marker for CD4+ T cells?

We want to identify a certain type of lymphoid cells in cancer tissue however for that we need to identify and exclude three different types of cells (T, B and Natural Killers) using immmuno-histochemistry separately applying antibodies (CD56, CD2 and CD 20) on separate )sections/slides by assuming there will not significant difference among four sections at 4µm thickness.

Brief procedure is to make 4µm thickness slides from the same tissue sample obtained from FFPE blocks, first slide will be exposed to CD2 to identify T cells, take photograph using light microscope and export the image to Fiji and identified T cells will be given a color,

Similarly the same procedure will be done on rest of three separate slides to identify B cells, NK and ILC 3 using rorgamma t antibody giving each cell type different colors. At the end we'll have 4 different Figi colored images.

Now the tricky part is how can we merge these four images together to get one single image with all four types of cells exhibiting different colors, so that in a single final merged image we can show all four types of cells WITH DIFFERENT COLORS.

We just incorporated a Northern Lights Spectral Flow Cytometer, and we want to analyze glucose incorporation in T lymphocytes using the 2-NBGD probe.  The thing is that we cannot find the spectral fingerprint (ribbon plot) from 2-NBDG. Anyone that could help us with this? And of course any information / advice you can give us will be deeply appreciated!

I'm interested in T cell subsets, and ideally, I would be able to capture some cytokine information. Has anyone tried directly injecting a mouse with Brefeldin A, without dissociating, isolating, and culturing T cells? (I always have such poor viability with the culture.)

I plan to check t cells from mouse tumors for IFN-y, TNF-a, IL-2 and granzyme B. Is T cell activation by PMA or anti-CD3/CD28 needed?

Thanks!

is there any traditional method to check T cell levels or can we isolate T-cells to check Th17 cell levels using specific markers? I am a little stuck here.

Just to clarify I don't want to use the magnetic separation method. I want to Try the traditional method before I jump to that.

Thank you in advance.

Hi,

Is there a way to analyze the VDJ regions that T cell falls into using the scRNA data? R package is preferable.

I am interested in isolating naïve CD4+ T cells from human PBMCs. I get whole blood (~500 mL blood + 70 mL anticoagulant) from a blood center but I don't receive it till +1 day after extraction. Blood sample is stored and delivered on ice. Once I get the sample, I start performing the Ficoll gradient. However, the amount of PBMCs that I have obtained (~0.25*10^6 cells/mL) is lower than expected (~10^6 cells/mL - this is the amount I used to obtain using fresh whole blood samples). I have been reading about keeping blood samples at room temperature before processing, does anyone have experience with this approach?

On the other hand, does anyone know about the percentage of naïve CD4+ T cells in the PBMCs fraction? Thanks!

Hello everyone,

I want to do an ELISA assay to detect IFN gamma and IL2 secreted from a primary T cell line.

In order to do so, on day 1 I will thaw these T cells and centrifuge them to get rid of the DMSO and then resuspend them in media consisting of : RPMI, FBS 10%, PenniStrep 1%, 0.5% non essentials amino acids, 1% HEPES, 0.5% L-Glutamine and 0.001% B-mercaptoethanol and 2nm of IL2.

Then on day 2 I will put those cells in 96 well plates with the APC that have been incubating with the antigen prior in the same media.

This incubation will last 48h at 37°C and then I will harvest the supernatant after centrifugation of the plate to perform an ELISA assay.

I read that feeder cells are important when working with primary cells but I don't know if I have to add them when working with such short time, if I don't put any will it prevent the T cells to activate and secrete IL2 and IFN gamma ?

Thanks in advance, I hope I was clear enough.

Best regards,

Sonia

Hi, guys

I have had big problem with Jurkat cell activation.

Protocol is as follow:

1. Seed jurkat at 1X 10^5 cell/ml and incubate for O/N

2. Treat jurkat with PHA(sigma, L1668-5MG) at 1, 10, and 50 μg/mL, and PMA (sigma, P1585-1MG) at 50, 100 ng/mL for 24, and 48 hours

3. Collect cell culture supernatants

4. IL-2 ELISA (abcam, ab270883)

But, IL-2 was not released from jurkat stimulated by PMA/PHA.

Instead of PHA, Jurkat was treated with PMA/ionomycin (invivogen), however, IL-2 secretion was not shown (PMA at 50 ng/ml, Ionomycin at 1 ug/ml).

I doubled the cell density, but it didn’t work out.

What's the matter with my experiments?

Is the cell in bad condition? Or Should I change the ELSIA kit?

Thank you

I want to detect markers of degranulation of T cells in HIV patients (mainly perforin and granzyme B), but I'm not sure if I need to stimulate the cells or not before test. Both are available in the published literature, what is the difference between them?

how do immune cells(NK cells, CD4 Th1, and CD8 cytotoxic T lymphocyte effector T cells) produce IFN-gamma? Is there a signal pathway?

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.

Hello, I am stimulating CD4+ genes with doxorubicin to see effect on FV, but am having real trouble finding a good endogenous control gene for qPCR. I have tried GAPDH, PMM1, 18S and HPRT1. None of these seem very good at all, 18S shows some promise but the others are no good.

I can't seem to find any research on this particular cell stimulation, and based on what I have found I am thinking of trying B- actin or RPLP0 and maybe combine these with 18S...

I am desperate for any kind of advice here.

Best regards, Kaja

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 indicat