T Lymphocytes - Science topic

As we know that the expression of PD 1 is less in jurkat cell. To express PD1 jurkat cell need to be stimulated or activated. But there is a chance of activation induced apoptosis. How this activation induced apoptosis can be reduced so that only cancer cell mediated apoptosis is highlighted. IS there any step to be followed after stimulation of T cell so that activation induced apoptosis is reduced?

I`m trying to observe pro-inflammatory cytokine release in T cells obtained from patient PBMC when exposed to a specific antigen in vitro.

But I`m not sure if I should use anti-CD3/CD28 or anti-CD28/CD49d because I saw in different papers that both can be used. I did read that CD3/CD28 is focused on T cell expansion while CD28/CD49d is focused toward cytokine amplification, but if I want to just check cytokine secretion would it matter which one I use?

Also would it be okay to use all 3 stimulatory factors (CD3, CD28, CD49d)?

Hi all,

I want to introduce two separate receptors in human T cells. I have the receptors in separate retroviruses and plan to follow a retronectin-spinoculation protocol. I was wondering if anyone had any experience with viral introduction of two separate genes and the best way to go about it? I haven't titred my virus as we can usually get around 40% transduction using fresh virus. Can I just do a 50/50 mix of the two viruses and then spin the plate to bind them? Is there a chance of viral interference or inhibition? They both have exactly the same backbone vector with a slight alteration to the receptor structure. Is it better to do it sequentially or simulatenously?

I will be perfoming a MACS sort but any advice on the actually transduction process would be really appreciated!

Thanks :)

Is the most significant breakthrough so far "Published in Nature Aging, the new research reveals that the trick to anti-ageing lies within the white blood cells known as T cells. The researchers behind the study discovered that they can reprogram these cells to turn them into ageing-cell-killing machines known as CAR (chimeric antigen receptor) T cells.

24 ene 2024 " ( https://www.sciencefocus.com/news/slow-ageing-t-cells ).

Hello!

We are using LNP to deliver circular mRNA into T cells. However, compared with linear mRNA, the transfection efficiency and protein expression duration are similar. Are LNP generation or transfection protocols different for circular mRNA delivery compared to linear mRNA. Thank you for your suggestion.

I want to study the effect of FoxP3- CD4 T cells on immune control of tumors in mice. Does anyone know a genetic (inducible-) mouse model where I can specifically deplete either all FoxP3- CD4 T cells or specifically Th1? If not, could you think of a way to target those cells specifically? Thanks in advance!

Is it somehow related to tumour microenvironment or some other reason behind this.

I use viral peptides to activate the T cells after isolating the PBMC. Is that enough to promote T cell expansion with interleukins?

Hi everyone,

I am seeking advice on the optimal protocol for RNA extraction from human T cells that have been frozen at -70°C. Specifically, would it be more beneficial to culture these cells for 18 to 24 hours in complete media (TexMACS + 10% human serum) with 100 IU of IL-2 before proceeding with RNA extraction, or should I initiate the extraction process immediately after thawing?

Any insights or experiences you could share would be greatly appreciated.

Thank you.

kindly refer the protocol of that experiment

I'm looking for protocols/sources that provide information about how to maximize the isolation of membrane bound proteins and general protein yield of unstimulated primary CD4+ T-cells. I have used RIPA and other ThermoScientific specialized protein lysis products and I have not succeeded.

These cells are isolated from fresh PBMCs.

My ultimate goal is to use Western Blot and ELISA as my protein assays.

Any suggestion is welcome. Thanks!

Some time ago, I wanted to perform immunofluorescence on the S protein of mouse CD4 T cells using a Leica super-resolution microscope. I fixed with 4% paraformaldehyde, then added it to the well plate that had been coated with 0.1mg/ml poly-L-lysine, and then proceeded with the routine IF operation. However, under the microscope, there were many fluorescent particles in the background, and different focal planes showed different results (for example, the S protein was inside the nucleus, and after adjusting the focal plane, it appeared around the nucleus).

I'm stimulating CD4+ T cells for 3 and 5 days with plate-bound CD3 antibody, soluble CD28 antibody, and LPS. I want to measure intracellular cytokine levels on days 3 and 5 (IL4, IFNγ, IL10, and IL17A) via intracellular staining and flow cytometry. When should I add brefeldin A to my cells in order to detect intracellular cytokines? 4 hours before analysis? 12 hours? Any help would be appreciated.

P.S. I've already analyzed T cells stimulated for 3 days for IL17A. I added brefeldin A 4 hours before analyzing the cells, but I didn't see any IL17A+ T cells. This is making me worry that perhaps I didn't add BFA add the optimal timepoint.

I saw kits for cell culture extracts but I didn't find any about assays directly with cells. Would it be possible to fix the T lymphocytes on the plate?

Hi, we are looking to activate in vivo mice reconstituted with OTI or OTII T-cells, and we are wondering if we should inject i.p or orally the OVA and with or without adjuvant. What's the best way to stimulate mucosal immunity? 

Hi! For reprogramming PBMCs into iPSCs, I understand that you first must obtain the PBMCs by a density gradient, such as Ficoll-Paque, and then you have a product of primarily lymphocytes with various cell types (T cells, B cells, etc.). I was wondering if it is known which cell types in particular are ultimately the ones that reprogram? And why during the expansion process of the PBMCs using the CytoTune kit do I not have to add some type of antibody to create T cell activation... or is that activation occurring from a component in the media that is proprietary and unknown? Hope this makes sense :)

I was asked this during my preliminary exam and I told the committee member that only cytokines are listed as an addition in the media, but no antibody-- this leads me to think that T cells are not the target for reprogramming. I've gone down a rabbit hole in the literature and only came out more confused.

Hi,

Does anyone have experience and success of isolating, culturing and stimulating hamster splenocytes for T-cell analysis (FACS and ELISPOT)? I am currently using a protocol that we routinely use for mouse splenocytes which gives good fresh and post-thaw cell viability (>70%).

Our mouse splenocytes are initially cultured and rested overnight in RPMI, 10% FBS, P/S, L-Glutamine and HEPES. For stimulation cells are resuspended in this media with the addition of IL-2 and Beta-mercaptoethanol and we have had no issues.

For hamster splenocytes however (using the same media and protocols for mouse splenocytes), we have been getting cell viabilities of around 50% which drops further after stimulating. Does a different culture media need to be used for hamster splenocytes? I found one paper that said RPMI was the best media, but haven't found any other publications that describe their isolation and culture of hamster splenocytes.

Any knowledge and tips on this would be gratefully received... Thanks!

Kind regards,

Lauren

I am attempting to culture tissue resident T cells (TRM) derived from fresh intestinal tissue for up to six days. I am finding that the viability of the T cells is low at D0, and a very small fraction of viably CD45+CD3+ cells remain at days 5 and 6 (less than 1% of all cells) as evaluated via flow cytometry. I conduct the same protocol on blood simultaneously, and viability in the blood is not an issue, and these T cells survive beautifully.

I did a double negative selection to remove epithelial cells, red blood cells, and granulocytes, and enrich for CD3+ cells. For my culture media, I use ImmunoCult T cell Expansion Media supplemented with PenStrep, FBS, IL-2, IL-7, and IL-15.

I am wondering if anyone has had success keeping TRM viable in culture? My next logical step will be to FACS sort for T cells rather than a bead enrichment, as I feel that dead cells and epithelial cells are still getting through the bead enrichment (this is supported by cells detected in my flow dump channel) and may be contributing to cell death. I am also considering adding TGF beta to my culture media. Any other suggestions would be appreciated!

Hi,

I'm analyzing some bulk RNAseq data from T cells under different conditions, and based on the RNAseq data, condition A shows higher expression of genes associated with effector functions compared to B; however, in my cell culture data, T cells under condition B showed superior functions.

In this case, how should I interpret the data as a whole story?

Thank you!

I want to detect the T cell differential when it co-culture with dendritic cells with the presence of OVA

In some research, immature BMDC co-culture anti-CD3、anti-CD28 stimulated naive CD4+T cell from wild type mice, and most of research use OT-II naive CD4+T cell, why OT-II naive CD4+T cell do not need stimulation of anti-CD3、anti-CD28 ?

Hi, scholars.

I am planning to conduct mouse neutrophil and T cell co-culture in Vitro and observe the effect of neutrophils on T cells.

I don't have any protocol. Could anyone provide me protocol and the reagents such as do I need to add any recombinant cytokines or peptide or anti-CD3 and CD28 to the culture medium. How long should I culture the T cells and neutrophils to T cell ratio.

Thank you.

Hello,

I am trying to transduce my CD4/CD8 T-cells with lentivector using retronectin from Takara (#T100B) at 50ug/ml, after they are activated by CD3/CD28 for 2 days.

The official protocol says that "it's important that the target cells be in logarithmic growth phase", but how this state may be monitored?

How to choose the "perfect moment" for transduction via retronectin?

Thanks in advance!

After PBMC isolation, we are not able to activate and expand T cells.

I am working on isolating Naive CD4+ T cells from human PBMCs using the Miltenyi AutoMacs. However, I am obtaining a higher percentage of Naive CD4+ T cells from the PBMCs than expected. I would like to confirm the identity of these isolated cells using transcriptional primers for mRNA gene expression analysis. Are there any specific primers that I can use for this confirmation?

Thanks!

Can I do flow cytometry analysis for CD3,4,8 positive T lymphocytes from the mouse spleen samples frozen in liquid nitrogen and stored at -80C?

Hi everyone

I produced a batch of virus , enriched it with centrifugation, and titrated it with T cells after 48h. For my experiments I want to use MOI 3 and that's equal to 1/12 dilution.

but at this dilution my cells will die after 5 days in cell culture( I use PBMC not sorted T cells), while untraduced control expanded 6 times during 5 days of stimulation. Until day 3 transduced cells are similar as untraduced cells. I tried up to 1/160 dilution but I still have very large cell death with this dilution!

Do you have any idea what is the reason for this?

Thanks

Hi everyone,

I'm going to generate CAR-T cells starting with mouse CD3+ T cells. I isolated mouse CD3+ T cells using Miltenyi Pan T cell isolation kit. I seeded cells 1millon/well in 24 well plate precoated with 1ug/ml antiCD3 and 2ug/ml antiCD28. Cells were culture in the presence of 50IU/ml rhIL-2. However, only 20% of the cells were alive after 48h culture. Not a lot of cells became bigger, and I didn't see clusters forming. My question is that is it because of not sufficient TCR stimulation or AICD? I'm using RPMI supplement with PS, Glutamax, sodium pyruvate, 10% FBS, b-Me. Thank you in advance!

Kindly guide me if any suitable protocols are there to study the interaction

Hi everyone,

I am staining leukocytes isolated from mouse kidneys in flow cytometry. I stimulated the cells with PMA/Ionomycin + BFA for 4 hrs in T-cell medium (RPMI + Pen/Strep + 10 % FCS + 50 uM beta-mercaptoethanol) and stained them for surface markers, T-cell transcription factors and IFNg and IL17A.

When I plot the cytokine production (IFNg-BV711 vs IL17A-BV650) in CD4+ T cells, I noticed that beside my single-positive populations, there are events on a somewhat straight diagonal line that seem to be double-positive. There are some other events that are double-positive that are more scattered around, which is why I think the events on the diagonal could be a technical artifact (see attached plot).

I am also attaching my FMOs for IFNg-BV711 and IL17A-BV650 where these events are not present.

I'd highly appreciate your thoughts on this.

Thanks a lot,

Jasper

I am looking at CD4 T cell populations in B6 mice at 42dpi with influenza B virus. I do perform CD45 IV labeling just before tissue harvest. Now I am running my samples on the 5L Cytek Aurora and I see this weird distribution of CD4 staining where it looks like there are two distinct populations of CD4 (see attached image). The CD4hi population (on the far right) is primarily IV protected and the CD4lo population is primarily IV labeled.

I also stain mLN, cLN, pLN and Spleen and they all look normal. This issue appears to be exclusive to the lung

Has anyone else seen this? I have been searching in the literature but haven't found anything helpful.

Thanks!

I need B3Z T cell hybridoma in my experiment. It is not commercially available. Can someone share some with me? Thanks very much!

Kindly, explain

Hello everyine,

Does anybody have a proof (a review or a scietific paper) that indicates if T cells from transgenic mices OT-1 and OT-2 express CD154 on their surface?

I couldnt find the answer on pubmed,

Thank you in advance,

What is the best serotype of AAV for infection/transduction of mouse or human T cells?

Dear All,

I've been working on differentiating monocytes to dendritic cells (DCs) in vitro using a 24-well TC-treated plate from Corning. Here's my current protocol: I place 2 Million PBMCs in each well, and after one day, when monocytes should be adherent, I carefully remove the supernatant and add RPMI with IL-4 and GM-CSF. After seven days, when I inspect the cells under a microscope, I observe that the Dendritics are not fully formed, and the cells appear smaller than expected. Additionally, they are not fully adherent; if I attempt to wash them, most of them detach. I also notice the presence of other immune cells, possibly alpha-beta T cells, which are similar in size.

I've attempted monocyte purification, but previous method seems to result in more dendritic cells.

Any suggestions, experiences, or recommendations on how I can optimize the purification and differentiation of my DCs would be greatly appreciated.

Thank you 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?

When we used LNP to deliver GFP mRNA to Jurkat cells, the efficiency was as high as 90%. However, when we delivered GFP mRNA to T cells, we failed, with an efficiency of 0.5-0.6%. Could anyone help us? Thanks a lot!

I used miltenyi whole blood CD3 micro beads to separate the T cells from whole human blood using the automacs seperator. Usually after the separation is done by the machine, there is a clear T cell isolate. Yesterday I ran the machine twice and the isolate had brown coloured sediments at the bottom both the times—probably micro beads. I have been using the same settings for a long period with no issues. Has anyone encountered this before and can someone please help troubleshoot? is this an issue with the beads or the column or something with the setting?

We are about to initiate #scRNAseq on some T-cell types. Back to literature, I found a wide diversity in the counts of the anlaysed cells used for the method (1K - 25K cells). I would really be happy to receive experts suggestions and hints regarding this problem.

I am looking into selecting T cells with microbeads for isolation and subsequent adoptive transfer in mice. Before labeling with microbeads, I usually block Fc receptors to ensure a more specific binding of antibodies. If I block Fc receptors, do magnetic selection, and then do adoptive transfer, will the fact that the Fc receptors are block affect anything in vivo (e.g. proper T cell function in vivo)?

Hi everyone!

I am performing an assay to evaluate CD107a production by CD4+ T cells using Flow Cytometry. Currently, I am stimulating a suspension of tonsillar cells with OKT3 only at 37°C for 3 hours. However, upon analyzing the results, I found that the signal from the unstimulated cells (basal) and stimulated cells are the same (attached is a picture of the corresponding histograms analyzed by FlowJo). Therefore, I think that the stimulation with OKT3 is not effective. I have read that cells can also be stimulated with CD28 in addition to OKT3. Does anyone have experience with this assay that can provide guidance?

Thank you

I would like to know whether B cell respond and bind to antigenic viral protein and further show through MHC which can produce different markers on each developmental stage of B and T cells?

I am immunophenotyping PBMCs by flow cytometry using markers for T cells to analyse the different subsets (Naive, CM, EM and EMRA), and to measure senescent and exhausted t cells. I want to evaluate the effects of 2 different interventions on these cells, so in order to do the statistical analysis to compare pre and post intervention values and between groups values, what parameter should I use? frequencies of each population? MFI?

Thank you!

We are interested in CD8+T lymphocytes form pancreas cancer patients. Is it possible to find a pancreatic cancer cell line with HLA class I molecules that could be recognised by CD8+T lymphocytes from an individual patient?

The treatment of A549 tumor cell lines by IFNb induces the expression of CMH-I and also PDL1. co-culture of A549 (pretreated with IFNb) with T cells leads to partial killing of A549. I was wondering if adding anti-PDL1 could increase the kiling of A549 cells by T cells.

Thanks for guidance

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).

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?

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

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.

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!