Science topic

CD8-Positive T-Lymphocytes - Science topic

A critical subpopulation of regulatory T-lymphocytes involved in MHC Class I-restricted interactions. They include both cytotoxic T-lymphocytes (T-LYMPHOCYTES, CYTOTOXIC) and CD8+ suppressor T-lymphocytes.
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I read several protocols about CD8 T cell culture, including using 1640 or aMEM with FBS or commercially established expasion medium, with IL-2 or IL-7 added and OKT3 activated. Their results show 100-1000 fold T cell expansion with high viability. But, following their protocol carefully, I only can expand my cells in a small number, with lots of cell fragments in the mudium. I can't find out why I cant repeat their results.
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Hey,
I meet the same problem, I only can expand my cells in a small number, with lots of cell fragments in the medium. Do you find the reason?
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Hello everyone. I am researching the influence of CD8+ T cells on macrophage polarization in vitro. But I don't know how to co-culture them, e.g. plates, cell concentration, culture time, etc. I think CD8+ T cells should be activated by CD3/CD28 before adding to the co-culture media. Do I also need to stimulate macrophages in advance? Should I use M-CSF or LPS+IFN-γ? What's the difference between macrophages derived from bone marrow and peritoneum? I appreciate it if anybody could give me some advice.
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Hi, I think you should activate macrophages to study the cross-talk between MAC and CD8+. For MAC culture you can use DMEM ( you can also use RPMI medium) supplemented with 10%FCS, 1%PSA and M-CSF ( 20ng/mL). On day 2 or 3 ( depending on the morphology of the cells) you can rinse and immerse the culture in fresh medium. ( working with primary cell cultures can be tricky, so to be sure that the cell culture would survive you should let the MACs attach to the culture vessel for more than 24h). Depending on the MAC phenotype that you would like to study you have to activate MAC culture with LPS+INF-γ ( 100 U/mL of INF-γ and 100 ng/mL of LPS) for the M1 phenotype and IL-4 ( 20ng/mL )for the M2 phenotype ( both polarizing states of MAC). For the co-culture, MAC should be grown to confluence on the bottom of the transwell co-culture chamber at a density of 5x104 .
PEMs ( peritoneal macrophages ) and BMDMs ( bone marrow-derived macrophages) are phenotypically distinct and have different expressions of lipid metabolism genes.
For more information on MAC co-culture systems and protocols you may refer to the papers attached below.
I hope you find this usefull! Good luck with your research !
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Hello everyone.
We isolated CD8+PD1+ T cells from the peripheral blood of a metastatic melanoma patient, with 99.5% isolation purity and 98% PD1 expression. We activated cells with Human TransACT "Miltenyi Biotec". After the activation phase had been finished, we left the cells in the expansion phase for 14 days, and then we performed a flow cytometry experiment to identify the phenotypic features of the expanded population. We found out that PD1 expression decreased from 97% to nearly 50%, but we still have CD8+PD1+ purity of 96%.
The part we didn't understand, what was the cause that led CD8+PD1+ T cells to decrease PD1 expression?
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PD-1 expression on naïve T cells is induced upon TCR activation. This transient expression decreases in absence of TCR signaling but is maintained upon chronic activation with a persisting epitope target. You used Human TransACT "Miltenyi Biotec", it may create a chronic activation on CD8+ Tcells. May be this activation results in lower expression of PD-1. Good luck
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Hi everyone, recently I've been struggling with the in vitro T cell activation assay, in which I incubated the PBMC with anti-CD3 and anti-CD28 (both concentrations are 2μg/ml) in the 96-well plate for 24 hours. BFA was added for the last 4 hours of incubation. After that, I performed flow cytometry to detect the IFNγ+CD3+CD8+ T cells. However, I cannot see the CD3+ T cells, and the stimulation effect was not so satisfactory (Just detected 5% IFNγ+ cells vs 15% in PMA/Ionomycin group). The CD3 antibodies for stimulation and surface staining are clone OKT3. I wonder if I can detect CD3 expression by flow cytometry after stimulation with anti-CD3/CD28, and how can I improve the efficacy of in vitro stimulation? Thanks a lot!
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Michael Förster Thanks for the detailed answer and suggestion! I will repeat the experiment with a different clone. I hope I can share a good result with you next time:)
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Hi everyone,
Could you please help to tell me how many kinds of subsets of CD8+ T cells? And what's the markers for subsets of CD8+ T Cells?
Thanks a lot!
Long
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Hi Michael,
Thanks a lot for your help.
Best,
Long
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if possible please elaborate the phenomena also
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Progenitor cells in the tumor are usually Granzyme K+ and slowly proliferate. As the cells flip towards transitory/effector-like they become Granzyme B+ and stay Granzyme B+ as they further exhaust. So in essence there are Ki-67+ (with the capacity to proliferate) cytolytic cells and non-proliferating/terminally exhausted subsets.
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I am interested in studying priming-associated gene expression in naive human CD8 T cells. I would like to find the best conditions for their short-term in vitro activation, so that their priming process is as close to physiological one as possible. I am planning to use plate-bound anti-CD3 and anti-CD28 antibodies. What is the concentration of anti-CD3 and anti-CD28 antibodies that you would recommend? Would you prefer soluble anti-CD28 to plate-bound one? Would you use IL-2?
Thank you!
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Dear Alexandra,
Thank you for your help!
I will check it out!
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Hi! I have cultured human CD34+ cord blood derived CD141+ dendritic cell (DC) using IL-4 and GM-CSF and have achieved around 30% CD141+ DCs. Now, I'm planning to stimulate these DCs with poly I:C (TLR3 agonist) and then proceed with co-culture with CD8+ T-cells. The literature available on the co-culture experiments are confusing to me. Could anybody please share any detailed standard protocol which is either available online or they have experience in DC-T cell co-culture experiments. Any help will be appreciated. Thanks in advance.
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Hello, @Paul David. Thanks for the information. I have used 20ng/ml of IL4 and 50ng/ml GM-CSF. I did not use any kit. Instead I sorted the CD11C+HLADR+CD141+CLEC9A+ DCs.
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Hi all,
I am culturing CFSE labeled OT-Ⅰ CD8+T cells with OVA peptide to stimulate cells for proliferation but it's not working well so far.
1) First, I pulsed the OT-Ⅰsplenocytes with 2ug/ml OVA peptide for 4 hours, then washed and resuspend in RPMI 1640 complete medium (with 10% FBS, 1% P/S , 2-ME, Sodium Pyruvate and NEAA) in plate.
2) After 24 hours, I isolated the CD8+T cells using a Miltenyi KIT and then labeled CD8+T cells with 2μM CFSE. I then cultured CFSE labeled CD8+T cells and added 1/5 CD8 negative cells(as APCs), 200ng/mL of OVA peptide and 50U of IL-2 to the wells.
3) After 16 hours, I check the viability using trypan blue and the cells dead 90%.
Any suggestions or comments on what I might be doing wrong?
Thank you!
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I think the problem is that that your CFSE concentration for staining is too high. CFSE is cytotoxic and kills cells. For that reason I would suggest a positive control with anti-CD3 and anti-CD28 agonistic stimulation as positive control.
Secondly, I would suggest to use this tried and tested protocol for CFSE staining. For the results, analysis and FACS gating, please consider:
Good luck with your experiment!
All the best & kind regards,
Michael
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Dear all,
we need a reference cell line for FACS experiments. We are looking for a human CD8+ T cells that
- we can culture easily and ideally forever, and
- has a defined TCR, i.e. it is known which HLA/Peptide complex it binds.
Any suggestion where to look are highly appreciated.
Thanks,
Clemens
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Hi,
I am wondering why our positively-selected CD8 T cells became two population on FSC-SSC plot (as shown below) after activation by using Dynabeads?
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click the square next to the color. so the yellow and red cells are the cells you want and green or blue cells are others.
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when dc cells incubate with tumor lysate, then co-cultured with CD8+T cells.
And the tumor lysate to act as an antigen, there has two methods to get the tumor lysate: one is using several cycles of frozen-thaw; another is using lysis buffer. and should i add protease inhibitors cocktail and pmsf when making tumor lysates?
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Interested
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Hi! I am culturing CD8+T cells from the spleen of infected mice, but they die a lot after ~16 hours.
Any recommendations (such as cytokines) to increase their survival?
IL-2 is not increasing it.
Thanks in advance
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I briefly wanted to reach out concerning your inquiry. Assuming that you are working with Trypanosoma cruzi infected mice, please try blocking the Fas/FasL pathway with a blocking antibody to overcome the apoptotic program and improve the survival of your CD8 T cells as per the attached publication.
I hope that helps.
All the best & good luck with your experiments,
Michael
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Hi, everyone, i am a fresher of tumor immunology.
Recently, i am preparing to study how pdl1 regulation in tumor cells will affect the function of t cells. and i have read a paper " miR-424(322) reverses chemoresistance via T-cell immune response activation by blocking the PD-L1 immune checkpoint. Nat Commun" . In this paper, author applied PBMC from healthy human with the anti-CD3, anti-CD28 and incubation of skov3 tumor lysates. Then, isolated CD8 T cells to co-culture with skov3 cells. i would like to apply this model, but i have a question, anti-CD3 and anti-CD28 has made CD8+ T cells activated, why do they need to add tumor lysates?
And, right now, i don't make sure whether i should apply antigen-specific CD8+T cells to coculture with tumor cells? other papers indicated that firstly isolated monocyte-derived DC and then make them matured with some cytokine stimulation and tumor lysates incubation. Then matured DC cocultured with CD8+ T cells to get the tumor-antigen specific T cells. These obtained CD8 T cells then cocultured with tumor cell lines. could you kindly give me some advice based on my research purpose?
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why do they need to add tumor lysates?
We need to add tumor lysates to optimize the ability of the cells to generate tumor-specific T-cell responses. Thus, cells can be loaded either with whole tumor cells or tumor cell lysate, or tumor antigen-enriched fractions to achieve the desired anti-tumor T-cell response. Tumor cell lysate represents the whole protein content of lysed tumor cells. So, the advantage of using tumor lysate is that the multiple antigens heterogeneously expressed on growing tumors can sensitize
T-cells.
DCs are the most effective type of antigen-presenting cells and can stimulate naive T lymphocytes to initiate an immune response. DCs represent the interface between the universe of foreign and tissue-specific antigens and T lymphocytes. They also play a role in all aspects of T-cell responses. I would suggest you use monocyte-derived DCs in your study as it will help in a better response.
Best Wishes.
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I was wondering whether CD8 T cells that are present in the spleen are also present in the para-aortic lymph nodes during atherosclerosis. I can not find any article that studies the specific cells (CD8 T cells that express granzyme K) in de para-aortic lymph nodes, and I thought that if the cells were present in the spleen, they might also be present in the para-aortic lymph nodes since they are both lymphoid organs.
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Yes, dear Fa Ro they can present in lymph node.
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I was wondering whether it is possible to turn off the expression of a transcription factor that is expressed by CD8 T cells, and if yes, how?
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Yes it us possible. You can design a sgRNA and then use CRISPR can system to knock out either the promoter region or the main gene of the transcription factor employing NHEJ mechanism of cells which is error prone and induces INDELs
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Anyone have explored CD3 - CD8+ T cells subsets in patho-physiological conditions ?
Please , share.
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My comment was deleted!
Yeah Rachel, actually after stimulation with PMA I faced that subpopulation in my interested cells, but of course, I had CD3+ within interested cells. Good to know one more tip in research.
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I would like to know about all the molecules that can be expressed by/in CD8 T cells
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Hi Fa,
Your question appears simple but there are several answers depending on the species (human vs mouse) and the state of the CD8 T cell activation.
Naive CD8+ T cells express CD62L (L-selectin) integrin, IL-7Ra (CD127), S1PR1 and CCR7 chemokine receptors.
They also express the costimulatory molecule CD28 and negative regulators of T cell activation such as TGFbRI and VISTA. These are all surface markers but they also express specific transcription factors such as Foxo-1 and KLF-2
Just after activation and during priming, CD8 T cells upregulate the expression of CTLA-4 and CD69 then later PD-1, and LAG-3 as well as Fas. As the CD8 T cells are primed for cytotoxic potential, they express Granzyme B (GzmB) and Perforin.
CD44 is an excellent marker for antigen experience of mouse T cells.
However, human CD8 T cells are different since naive cells are CD45RA+ CD45RO- while effector T cells are CD45RO+ CD45RA-
However, there are many more molecules than those I just listed. For a more comprehensive knowledge of these, please refer to these resources:
2-ImmGen is also a terrific resource where you can check for the expression of specific markers
There is one minor, yet important point to consider. The classically defined CD8+ T cell subset is the αβ CD8 T cell. However, there are also αα CD8 T cells which are part of intraepithelial T cells in the intestines.These cells do not express the β TCR subunit and their role in gut homeostasis remains unclear.
I hope this reply was of any help.
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CD4 T cell gets activated when the pathogen is being presented through MHC-class II ,while the CD8 gets activated when the pathogen presented through MHC-class I.
How these two get activated in every infection??
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Hi,
I am agreed with Lora Benoit. There are differences in the mechanisms regulating the generation and maintenance of CD4+ and CD8+ effector and memory T cells.For both CD4+ and CD8+ T cells, transient exposure to antigen is sufficient to induce an antigen-independent program of proliferation and differentiation.After being activated, naive CD4+ T cells differentiate into functional subsets called T helper type 1 and T helper type 2 cells, based on their production of cytokine IFN-γ and IL-4, respectively1. T helper type 1 cells are essential for protection against a variety of intracellular infections, whereas T helper type 2 responses can be protective against certain extracellular infections. CD8+ T cells mediate their effector functions through production of cytokines such as IFN-γ and tumor TNF-α and/or by cytolytic mechanisms.
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I recently did some experiments with the drugs I am working on and found that after treatment, although we see an increase in CD8+/Treg ratio, the percentage of IFN-gamma secreting CD8+ T cells decline drastically. Can other cytokines like IL-2 or TNF-alpha activate T cell effector function in the absence of IFN-gamma?
I am not an immunologist and I could not find any reasonable explanation for this either. Almost all studies I referred to showed that IFN-gamma levels increase when there is an increase in the proportion of CD8+ T cells.
Any help in making sense of this is appreciated.
Thank you!
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Dear Amirali,
Usually, as you said, IFN-g is an important marker for intratumural CD8 activation. However you could check other parameters:
1) granzyme B, CD69, PD-1 and other checkpoints on T cells. Maybe you have more T cells but they are anergic/ineffective, due to immunesuppression.
2) I assume you are doing this experiment from mice tumors. If you still have cells frozen you could also check for MDSC and NK cells. If your tumor has low expression of MHC I, CD8 cells could struggle to detect it and get fully activated. However, NK could do the work in this scenario rather than CD8 T cells.
In addition, you could repeat the experiment to be 100% sure that you see this decline in IFN-g and it is not dependent on your staining procedure (maybe something went wrong with the staining and you see few cells IFN-g+)
I hope this helps.
Regards
Francesco
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For an adoptive transfer would like to isolate CD8 T cells that express granzyme K and EOMES from the spleen. Is it possible to first isolate CD8 T cells with an isolation kit, followed by the use of antibodies for granzyme K and EOMES?
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If you're worried about antibodies sticking to your cells after isolation, I can recommend the Fab-TACS® system. Instead of high affinity antibodies, low affinity Fab fragments are used to positively select cells. They completely dissociate from the surface after isolation, so you don't have to worry about antibodies remaining on your cells. You can check out the products here:
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I want to visualize EOMES, a transcription factor, in CD8 T cells. I am planning to perform an immunofluorescence test and I was wondering what kind of stain or chemicals I can use to enable the visualization of internal markers of a cell.
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Fa Ro , that does make this quite a bit more complicated.
Fluorescently-conjugated small peptides (i.e. phalloidin) can be used to visualize some intracellular targets in live cells (actin filaments in the case of phalloidin), but this does not apply to most proteins. Unfortunately the size of normal antibodies prevents their entry into live cells, but a few different approaches for labeling intracellular targets in live cells (e.g. quantum dots and nanobodies) have been published. Unfortunately, none of these systems are in widespread use.
The most common approach for live cell visualization of a specific protein is constructing a fluorescent fusion protein. This can be done with transient transfection or through generation of stable cell lines. However, there is no guarantee that the fusion proteins will behave in the same manner as the wild-type due to steric hindrance.
One group has already reported a GFP-Eomes fusion protein in mice: . They used TALENs to insert the GFP into the first exon of Eomes, but you should be able to reconstruct this in a plasmid.
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I have been looking at all kinds of articles but it is not very clear for me. CD4+ and CD8+ T cells have different functions. However, they do both secrete IFNy. So is the function of IFNy secreted by both T cells the same?
Can IFNy secreted by CD8+ T cells only induce apoptosis? And if so, how?
Can someone tell me in detail how IFNy from CD8+ T cells exert actions?
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IFNg itself is the same protein regardless of the producing cells, but its effects are context-dependent.
IFNg from DC will stimulate CD8 T, for example.
IFNg from CD8 T will prepare fibroblast for apoptosis in case viral infection, etc.
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Hi!! Do anyone know if CD8+ T cells are affected by CpG in a culture? Because I want to coculture B cells and CD8+T cells, so I have to activate B cells with CpG; but what about CD8+T cells and CpG?
Thanks!!
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Dear Yamila Gazzoni Yes, CpG does effect the clonal expansion of CD8 cytotoxic T cells. I hope the publication attached will be helpful in answering your query as the stimulants used in B cell culture (B Reg differentiation).
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For my project, I need to use CD8+ T cells and follow exhaustion protocol. There are usually kits available with negative selection to obtain untouched CD8+ T cells.
Due to the way my experiment is planned, I need to use the fraction that is not T CD8 + T cells to isolate another cell type in the same patient, which would already be labeled after isolating CD8+ T cells. Unfortunately because of all the steps I have to go through to get the other cells, I could not use previously labeled cells.
I wonder if anyone has used positively selected (labeled) CD8+ T cells to do the exhaustion protocol with good results (I am going to check for exhaustion markers after the stimulation). I have searched papers and they all use untouched CD8+ T cells.
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Hi Valeria,
I have done flow cytometry, functional assays, and single-cell sequencing with CD8+ positively selected exhausted T cells. In my case, negative selection is not a good option for tumor samples. Cheers.
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in vitro mice CD8 cell activation. These cells are extracted from B6 mice spleen by magnetic separation.
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CD3 CD28 beads
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I am trying to isolate gDNA from sorted CD4 and CD8 T cells. Human PBMCs will be thawed stained and sorted on a FACS Aria. DNA will be isolated immediately after sorting.
What yield of DNA can I expect if I start with 500k sorted CD4 or CD8 T cells?
Some preliminary experiments have given me less than 1ug of gDNA starting with 500k cells.
I am planning on trying out the NEB Monarch DNA isolation kit, PureLink Genomic DNA Mini Kit and the QIAamp DNA Micro Kit. Any suggestions on which kit is better suited to use with lower cell numbers?
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DNA extraction from T lymphocyte yield increased according to the cell numbers,number_dependent manner,so 100 ml buffy coat yields~300 million pan T cells has been widely as fesible approach to deliver DNA.the same idea used to storte thr extract of DNA on rnnning on a FACS Aria the isolation.N.B differnt protocol applied accoding to the source T lymhocyte.Thanks
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There are various points of view some of them reported the ratio of early/late apoptosis as killed tumor cells by CTLs. Others reported the sum of early and late. However, we know CTLs kill tumor cells through lysing so it seems PI positive should be considered as the killed cells. What is your idea?
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Dear Lora, thanks for precise answer I have co-cultured colon cancer cell line with splenocytes of mice for 24h. So the rate of death cells were considerable relative to the control. I've read your answer in some articles. Ok I will evaluate the %Anx and %double positive. So what should I do. I mean next I should report the ratio of %Anx/%death cells? This is an article which is associated in this case but at the end I got confused how the results were reported I attached it for you@ Lora Benoit
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I work with mouse OT-I CD8+ T cells which have been activated in vitro with peptide-MHC and B7 proteins for 3 days. I've been trying to standardize protocols to fix these cells using paraformaldehyde (PFA). I usually add equal volume of 2x concentrated PFA to achieve a final concentration of 3.5% PFA to the cells, and fix these at 37C for 10 minutes. The PFA I use is diluted from a 16% PFA stock (aqueous solution from Electron Microscopy Sciences, catalog# 15710) in 1X PBS. After the PFA incubation is complete, I wash the sample thrice with 1X PBS, with 5 minutes between each wash.
When I viewed the cells fixed using the above protocol using Interference Reflection Microscopy, the cells seemed to have "cavities" or "pits" (sample image attached). Does anyone know what causes these "cavities" and how can I modify my protocol to prevent these from occurring? Fixation of OT-I T cells using 4% PFA for 10-15 minutes seems to be used pretty commonly in the literature, so I'm not sure what's going on.
I would really appreciate any suggestions regarding this! Thanks!
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I would recommend storing the stock solution at -20 and ensue minimal contact with light. That said, I am not sure this is entirely artifact that you are observing. As per your protocol, these cells have been activated on siinfekl. I would expect granules containing perforin and granzyme at this time point.
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Such a thing should be hypothetically possible as B-Cells like all other nucleated cells possess MHC-I, & might be able to cross present extracellular antigens similar to other professional APC's via MHC-I to cognate CD8+ T-cells. Does this process lead to the cytolytic killing of the antigen presenting B-cell by cognate CD8+ effector/central memory/antigen primed T-cells in the lymph node? (If so what are it's implications w.r.t the adaptive immune response?)
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It is a normal part of the contraction phase of the immune response
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Hello everyone,
I'm currently developping a topical treatment and studying its biological effect for inflammatory dermatoses.
I would like to study its immunosuppressive effect on skin T cells from mild to moderate psoriasic patients. I had thought of topically applying the treatment on the epidermis of the biopsies for a predetermined time, then isolate T cells and study their phenotype, but the low surface area of biopsies (about 4x4mm) makes it a little bit difficult espacially since the treatment looks more like a thick milk rather than a cream, it would then fall in the culture medium which I do not want. I would like the formulation to suppress T cells activity by penetrating from the top of the epidermis, and not from the biopsie sides.
What would you do ?
And, supposing we could find a solution to my first problem, how would you study skin T cells ? I first thought about flow cytometry, but it would require quite a large amount of cells from such small biopsies.
I also thought about isolating T cells from the epidermis, incubating the cells with PMA/Iono and Brefeldin A then mRNA extraction + RTqPCR to quantify cytokine expression. Would that be okay ?
Kind regards,
Maxime Sintès,
PhD Student
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With such a small biopsy, I would use IF microscopy on 1/2 of each biopsy to stain for cd3 and th1 v th2 v th17 cytokine signal co-localization.
I would then do qpcr on mrna extracts on the remainder of 3ach biopsy for all three cytokine families and tcr alpha beta and tcr gamma delta and then normalize the data.
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Hi Everyone,
I am trying to optimize ChIP for sorted CD8 T cell obtained from mouse splenocytes(primary cells). I am doing the ChIP with a kit from Cell Signaling Technologies[SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003]. I kept 5 million sorted naive CD8 T cell for activation and after 3 days I got 8.5 million activated CD8 T cell. I did the entire process following the protocol provided in the kit word by word. it was mentioned in the provided protocol to use 0.5 μl Micrococcal nuclease for 20 mins and sonicate with three 20 sec pulses on bath sonicator with 30 sec gap between pulses which yeilded me 81ng/ μl of DNA after purification. but when I ran the sample on 1% DNA gel I found that my DNA sample has been overdigestedsince it was mentioned that DNA fragment size need to be in between 150bp to 900 bp for ChIP.
I looked for papers regarding this thing but found none that used the kit for primary cells.
Can anyone give me any suggestion that I can use for my next set of experiments?
should I reduce the Micrococcal nuclease digestion time or concentration? or should I use less pulses instead?
I am attaching the photo of my gel and the document portions in this question.
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Hi Anwesha. As Joshua Beytebiere already mentioned, using both sonication and MNase digestion on one sample is rather unusual. Nevertheless you are correct with the assumption that your chromatin might be overdigested if you want to go for 150-900bp, assuming the lowest marker band is 100bp. Both MNase and sonication treatment can be altered to achieve this as you already pointed out, but which to choose propably comes down to what you are trying to achieve. Sonication is considered more "rough", but less biased while MNase digest is more "gentle" but slightly more biased. Additionally, what protein is your target in this ChIP? This might be a key factor on which method you should focus on.
Sadly, I don't have a lot of expierence on sonication so far, but from what I have read Joshua Beytebiere is very experienced in this field and should be able to pinpoint what exactly you should tweak. As for MNase digest, many factors will determine your final outcome (time, temperature, concentration of both MNase and chromatin, the MNase you are using, Ca2+ availability, ...). Personally, I would recommend to stick to 37°C, 10min (optimal with shaking at approx. 500rpm) and do a titration of your MNase with aliqouts of the same starting material and varying MNase concentrations. Afterwards, do the general RNase, Proteinase K and purification steps and analyze by agarose gel. If you want to go for smaller fragments (say 300bp or so) I would also recommend to use a 1.5% agarose gel for a better resolution of smaller fragments.
I hope this might help you.
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Hi, everyone:
I want to determine whether IL-2 and IL-15 have different effects on immunized CD8+ T cell effector function.
In this study, I immunize WT mice with peptide on day 0 and 7, and harvest spleen on day 14 post immunization.
For investigation of cytokine effects, I purpose to re-stimulate splenocytes with peptide and subsequently treat with IL-2/IL-15 in vitro, and analysis of IFN-γ expression via intracellular staining.
Does anyone have any recommendation for in vitro stimulation protocol?
Detailed methods such as time duration of peptide re-stimulation and cytokine treatment? What time points should I harvest cells for analysis?
Look forward to your replies!
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Hi!
I performed LCMV infection model. There, everything was easy and straightforward. Day 8 post infection, you collect splenocytes and add the peptide of interest (1 mcM is a good concentration to start with) together with BfA from Biolegend. 6 hours later, you just stain live/dead and surface markers, fix, permeabilize and stain with anti-IFNg.
I used 5-6 millions splenocytes per round-bottom 96-well plate, because I required a rare CD8 T-cell population and the cell losses during washing steps are just horrible. Probably, you will face the same problem.
I haven`t tried cytokine stimulation along with peptides, but in my case, I believe, it wouldn`t affect the outcome significantly.
If you plan to culture splenocytes with peptides for several to expand your cells then the situation will be slightly different .
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Hi all, I'm doing some research about immunotherapy. I have some question about tetramer staining.
1. Should I use CD8, CD8a or CD8CD3 to lable mouse T cells?
2. Some researchers use CD16/32 to treat before tetramer staining while others don't, is that necessary?
3. Which sample is better to use, blood cells or splenocytes?
Thanks for your answer!
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1. Cd3 and cd8 needed
2. Yes, use fc block
3. Not sure what your model is or your time points to assess, without this information, I would say blood.
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When looking to establish Ag-specific CD8 cytotoxic T cells with autologous dendritic cells, there are numerous tumor-associated Ags to choose from. Does anybody know which Ags give the best results in terms of % of responsive CD8 T cells (e.g. gp100, tyrosinase, Melan-A... any others??) Are there great differences between these Ags in the frequency of potentially responsive CD8 T cells??
Thank you!
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Question: When looking to establish Ag-specific CD8 cytotoxic T cells with autologous dendritic cells, there are numerous tumor-associated Ags to choose from. It depends on what the tumor is that you are targeting. My preference is for the intact antigen/sequence and using an Adenovirus vector to infect the DCs. This works for stimulation and then for targets I like to use a pox vector infected target.
If you just want to get a T-cell response to an antigen I would use tetanus or as a second choice flu. Vaccine and antigens are easy to obtain.
The more critical question is what assay to use to measure outcomes. This is especially critical for tumor antigens, which following immunization may have a T-cell clonal frequency of 1 in 50,000. I like to isolate CD8 T-cells and use an ELISPOT assay.
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Hello, I have used SEB for T cell stimulation but SEB binds to MHC II and TCR, why is it so potent to simulate purified T cells not PBMC without any APC in it? T cells do not express MHC II right? Anything explained that I missed? Thank you in advance. I use human T cells from PBMC, not mice.
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yes. SEB is binding the Vbeta of one T cell to the MHC-II on another T cell.
Probably you were taught that only macrophages, DC and B cells express MHC-II, as well as endothelium. Paradigms are taught in class, there is a body of literature from 20+ years ago that addresses MHC-II on human T cells. A good paper that examines the mechanics of this using primary human T cells and Jurkats can be found: https://www.jimmunol.org/content/168/2/763
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Hi,
does anyone know if the level of HLA class I expression on CD4 or CD8 T cells changes upon T cell activation?
thanks
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Hi Becca :) We have a scRNAseq-dataset of human T cells pre&post activation. I checked whether I find more HLA-A/B/C RNA in the activated cells.
There is more HLA-A/B/C RNA, but this is probably just because activated T cells contain more RNA in general. After normalization for library size, the counts-per-million of the HLA-A/B/C RNA remain the same post activation.
Of course you'd rather want to check the protein itself but this does at least give some info I hope
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When preforming ELISA on supernatans from PBMCs and spleen in mice, I saw that the blood generally had a stronger CD8 reponse in comparison to the spleen (i.e higher IFNy levels). However, this was not the case for CD4 T-cells after immunsation with a CD4-inducing vaccine?
Is this a normal for CD8 t-cells? Do memory CD8 t-cell circulate more in the blood compare to the spleen?
Many thanks,
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Dmytro Shytikov I took the blood from the heart and did a density gradient centrifugation using Lympholyte as separation media.
I hear you, I might have to adjust for CD8 T-cells in both sites then :)
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How can we assess the presence and amount of T-cells in the freshly removed skin tissue after surgery or punch biopsy? Also, if we freeze the skin tissue at -20C for a long time, after thawing to warm PBS or water, how can we make sure we have T-cells especially CD4 and CD8 T cells in the extracted frozen skin tissue? Does skin have to go in a specific solution or it has to be assessed for the specific markers ?
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Attaching papers for your reference. It is quite common to look for lymphocytes (CD4,CD8) from fresh punch biopsy. Not much knowledge about frozen biopsies.
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I have six samples, 3 replicates of "Naive CD8 T cells" and 3 replicates of "DMSO-activated T cells". I am doing an RNA-seq differential gene expression analysis to compare the expression levels of genes between Naive CD8+ cells and activated CD8+ cells, and produced this PCA plot in R. Can you interpret what this plot is saying about my samples?
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Hi Lilia,
first of all the PCA mainly shows what you would expect: the principle component 1 discriminates between the conditions (naiive/activated). Furthermore, samples are separated by some measure in PC2 in which it seems like there is a connection between one replicate each of the two conditions. The easiest explanation would be some "systematic bias" in the samples, for example the lower two points are from donor 1, the middle from donor 2 and the upper ones from donor 3.
PC2 explains 19% of the variance, which means it is a much smaller contributor compared to PC1 (57%).
A handy explanation of PCA plots can be found here: https://setosa.io/ev/principal-component-analysis/
Hope this helps!
Alexander
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Hello, Does anyone have any experience with isolation of CD8+ cytotoxic T cells and natural killer cells from rat spleen? I'd prefer kit-based if possible, but any protocols you can provide would be helpful. I am fairly new to cell isolation, and all the kits I am coming across are mice-based. I see this kit for CD8+ T cell isolation from StemCell ( https://www.stemcell.com/easysep-rat-cd8-t-cell-isolation-kit.html) but I don't see many citations for the rat version of the kit. I can't find any kits for NK cell isolation from rat spleen.
Anything you could tell me about the process (best practices, things to avoid, successful kits and techniques) would be very helpful.
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Hannah, in a single tube you should add CD3,CD8,CD4 and CD16/56. On FACSAria you may collect 3-4 population (NK cells as well as Cytotoxic cells). You may gate accordingly; CD+CD8+CD4- for Cytotoxic t cells and CD3-CD56+ for NK (use respective markers of rat).
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Hi all,
I need to work with CD8 Effector T cell and PD1+ CD8+ T cell. Does anyone know which cell line I can use? I have found out TALL-104 is human T cell but I don't know whether it expresses PD1 or not. For CD8 Effector T cell, I guessed I have to culture TALL-104 with some stimulator?
Can someone help me?
Thank you so much!
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I see now MOLT-4 is a CD4 cell line.. Did you find a good cell line in the end? I'm also interested!
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As you may know, blocking the receptor binding domain (RBD) of novel coronavirus could be very effective to prevent more severe effects of the inflammation. So, can a BsAb co-targeting CD8+ t cells and recombinant RBD protein (S1 or S2) of SARS-CoV-2 be sufficient to generate a good population of memory CD8 T cells against the future exposes to the virus?
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Very interesting
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In trying to getting more effective neoantigens for cancer therapy, I have some questions as followings.
1. T-cell activation is considered to be more important than B-cell activation. Which type of T-cell is more impotrtantbetween CD8+ T-cell and CD4+ T-cell? And what is the reason?
2. If a neoantigen have an activity for B-cell activation (i.e. for antibody production), does it have also the activity for T-cell activation?
If not, it is considered there are no correlation between them. Is it right?
Your answers would be very appreciated.
from DM
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1. Between CD4 or CD8, most would say CD8. First, CD8 recognize MHCI presented antigens, which all cells can present, thus they can have direct cancer cell activity. Also, most CD8s are cytotoxic, and can kill targets. CD4 is restricted to MHC II presented antigens and mostly are helper Ts. They direct immune response, but may not interact directly with targets as easily as CD8s.
2. B cell activation and antibody production isn't exactly without T cell help. Unless the antigen is strong enough to induce B cells to isotype switch without CD4 T cells... antibody production is still dependent on CD4 T cell recognition of antigen.
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What I usually do to activate CD8 T cell is culturing 10^6 splenocyte in RPMI with 5ug/ml anti-CD3 for 48 hours. Now I want to have effector T cell, I intend to do in the same way, but after 48 hours, I will collect the cells then change to new medium adding 2ng/ml rIL-2 (Biolegend). After 48 hours, I will collect the cells. Do you think this protocol works?
Another thing is I need to test effector T cell with a drug, so I intend to add the drug with rIL-2 at the same time.
I really need your help!
Thank you so much!
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My preference is to use anti-CD3 and anti-CD28 beads for human cells. We are culturing with IL7, IL15 and IL21 to address exhaustion. Again this is with human spleen cells.
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Procedure: Plate bound CD3 (10ug/ml) and soluble CD28 (2ug/ml) with 50u/ml of mouse IL-2
Started with 5*e6 and ended with less than ~2*e6 !
Could visually observe increase in cell # and also confirm CD8 activation flow (~70% were Tcm). However majority of the cells stained partially with trypan blue during cell count. Could anyone help me troubleshoot this activation procedure ?
Thanks
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Thank you @ Michael Förster
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Hi
I am trying to labelled murine CD8 LT by immunofluorescence on thymus and tumor sections embedded in paraffin and dewaxed. I meet some difficulties. I tried several Antobodies without success. Someone would have experience in the field and could help me by giving me the references of a working Ac and specify the protocol if it contains a particular point. Thank you very much.
Yannick
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Hi Yannick, I had a good experience with Mouse-on-Mouse (M.O.M) Basic kit (from Vector Laboratories) some time ago when the only good antibody available was on raised in mouse and I worked on murine material. I have checked on their website and the sample microphotographs are IF with virtually no background. It is a ABC type kit containing a mouse IgG blocking agent.
To have IF detection you would need to use fluorochrome-labelled avidin or streptavidin as a last step.
GeneTex has a convincing microphotograph on the spec sheet for their mouse anti-CD8 [LT8] antibody with DAB as chromogen. You have a good chance to use this Ab, or another mouse Ab, the Vector kit and AV or Strep/fluorescent tag.
Good luck with your project.
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Hello dear colleagues
I plan to test the primary antiviral response of CD8 T-cells of different genotypes, WT and with total knockout of some gene. The problem is that the KO mice that I plan to use have defective NK-cells as well, so I`m not quite sure that the effect I`ve got are exactly because of CD8 T-cells but not influenced by defective NK-cells.
So I want to purify CD8 T-cells from the mice of different genotypes and inject them into sub-lethaly irradiated WT mice (550 rad) and infect these mice with LCMV.
The question is: how should I treat these mice? Are there any special peculiarities in their maintenance? Can they survive maintenance in non-SPF conditions for ~8-10 days?
Many thanks!
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As I tried it, nothing special. Autoclaved acidified water with antibiotics, and everything works fine even in far non-SPF conditions. At least for the short term, 2-3 weeks.
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Hello!
I plan to check the protective effect of the adoptive transfer of some memory CD8 T-cells against the LCMV infection. So I think to establish LCMV meningitis model and to see whether the mice which have got CD8 T-cells from the LCMV-immune mice will be protected against LCMV meningitis.
In the Current Protocols in Immunology, 2001, M von Herrath and J L Whitton stated that I need to use 1 ml 27G syringes and anesthesia for intracerebral injections (like sedate mice and make a careful injection 5 mm dorsal from the eyes).
Do I need any additional equipment, like stereotaxis, Hamilton syringe or anything else or small insulin syringe is enough?
Do I need to perform LD50 titration or I can just use some sufficient viral titer, let`s say 50-100 pfu/mouse?
I`d be thankful for any tips or advices.
Many thanks!
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BTW, I`ve established the working protocol for this model, so can help if needed.
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I want to check the effect of IL2 on the phenotype of a population of mouse CD8 T-cells I'm studying. So I want to stimulate my cells with IL2, anti-CD3 or anti-CD3+IL2. Recently I've ordered 100 mcg human recombinant IL2 from Peprotech.
In the manual it's suggested to dissolve powder in 100 mM sterile acetic acid as 1 mg/ml. I equilibrated temperature of the vial, centrifuged, dissolved in 0.1 M acetic acid, waited for 3-5 minutes to give better solubilization. Then I dissolved my protein solution additionally to 50 mcg/ml in sterile PBS/0.1% BSA, aliquoted and froze at -80. 1 vial was diluted more in order to get 20000 U/ml as the instruction claimed IL2 activity to be 10 millions U/mg. So as I got 100 mcg IL2 it means I got 1 million units IL2.
But this IL2 added to get 50 U/ml doesn't support CD8 T-cell survival neither alone nor during polyclonal T-cell stimulation and even decrease their survival what is strange.
Why it is so? Any ideas? Any suggestions?
Many thanks!
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Cell stimulation protocol is like that:
Sorted CD8 T-cells are either cultured at IL2 50 U/ml or at plate-bound anti-CD3+soluble anti-CD28 or at plate-bound anti-CD3+soluble anti-CD28+IL2 50 U/ml.
As I tried before IL2 induced my CD8 T-cells to proliferate even at 25 U/ml.
Now it is not only supportive alone but it even reduces anti-CD3 induced proliferation.
Any idea where the mistake can be?
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I am currently working with RMA/S cells and found a few old literature on them. They are growing like crazy and Im not sure if its their natural growth rate. I would like to know about their required media, their doubling time, and the best way to infect them for expressing a protein. Thank you so much
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Hi! Mahboubeh,
I am looking for these cells RMA-S. Would it be possible for you to send a vial?
Please let me know. My email is pujag@stanford.edu
Thanks,
Puja
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Hi, everyone!
I tried to do mouse MDSC and T cell coculture assay, but failed. I have some questions.
1.Bone marrow derived MDSC can inhibit non-specific activated CD8+ T cell or not? (CD3 and CD28 activate)
2.I tried to isolate MDSC from 4T1 tumor using BD INFLUX. But the percentage of MDSC in tumor is too low to get enough cells, only 2-3%.
Which tumor model do you use, and how do you isolate MDSC from tumor.
Are there any flow cytometry better and faster than BD INFLUX?
Thank you!
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Hi Xin,
First off MDSC assays can be tricky based on the basic phenotypes you might be using for monocytic (CD11b+ Ly6c+ Ly6g- or CD11b+ Gr-1 int) vs granulocytic (CD11b+ Ly6c+ Ly6g+ or CD11b+ Gr-1 high) MDSCs that are also the same markers you would use to identify monocytes and neutrophils, respectively. Because of this, you should be aware endogenous MDSCs are a mixed population of all these myeloid cells and you’re really looking at the net effect of the population as a whole.
1) Yes, CD3/CD28 agonism-induced T cells can be suppressed by MDSCs but the extent depends on your readout. These cells suppress but do not completely impair T cell proliferation/function, so you would probably not notice big difference in proliferation by Ki-67 compared to something like CSFE dilution. Likewise, a subsequent T cell killing assay would be more sensitive assay compared to an INFy stain.
2) Recovery of immune cells can be difficult in the 4T1 model if you allow your tumor to progress to the point where most of the tumor is dead fibrotic tissue. If you’re seeing ulcerations on your mouse, try using an earlier time point for recovery from a 4T1 tumor. MDSCs might actually be that rare if you’re recovering them from the primary tumor; however, in theory they are a systemic population that you can recover from alternative organs (e.g., spleen). This would most definitely affect the ratios of MDSCs vs monocytes/neutrophils this population would represent.
3) In the past I have used Gr-1 column enrichment from splenocytes recovered from a 4T1-bearing mouse. These populations did show suppression but have to be interpreted with the aforementioned caveats. Alternatively, you might try negative selection for Ly6c followed by positive selection of Ly6g (or FACS sorting) if you want to parse out monocytic vs granulocytic suppression.
Hope this helps and good luck with your experiments.
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Does anybody know of any papers that address this question? I know CD8s do express 2,3 sialic acid which is recognized by at least the bird flus:
so in theory flu should be able to enter the CD8 cells, but I'm not sure if that has actually been observed/reported and a Google search only returns papers related to CD8-mediated response to flu, but maybe I'm just using the wrong keywords.
I'd appreciate any help/pointers you may offer!
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I phenotyped CD8+T cells isolated from healthy donor PBMC and stained them using anti-CCR and anti-CD45Ra antibodies to evaluate their differntiation state before and 2 days post activation of these cells using anti-CD28 and OKT3 antibodies?
I recovered more naïves cells (43%)2 days post activation than before (11%), does anyone can explain that?
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it is % of the CD3+CD8+ population
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For example, mice are infected with acute LCMV virus. Normally, CD8 T expand and reach effector function at day 7 when virus was cleared. I am wondering what happen if mice are reinfected with virus at day 7 post primary infection ? Does the effector CD8 T cells clear virus more rapid or these effector CD8T cells start activation induced death or become anergy due to re-exposure to strong antigen? Thanks
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If the re-infection is done with acute LCMV . The virus will be cleared.
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As I know, Jurket E6.1 is naive T cell, so we need to stimulate them to become CD8 or CD4T cell.
Does anyone know which stimulator I should used to differentiate Jurkat E6.1 cell to become effector CD8 T cell, or CD4 T cell?
I am a master student and I felt very lost about this. The previous person treated this cell with PMA to produce IL-6, IL-10. If want to see TNF-a, treating with PHA. She does not care about which cell it become, so that I am very confused.
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I'm pretty sure Jurkat cells are CD4 (not CD8s). Naïve T cells are either CD4 or CD8, they do not switch from one to the other (at least not that I know of).
PMA or any of the other plant based mitogens do not differentiate, they just activate. For PHA, you will need Ionomycin.
They are NOT technically naïve and as a cell line, they are limited to how much your can push them to the various t cell helper subtypes.
Depending on your goal, it might be better to use primary cells. I'm not sure you can get Jurkat cells to produce TNFa.
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I'd like to infect an immortalized mast cell line with a virus, incubate for a day or two and then co-culture with purified CD8 T cells to induce antigen-specific activation. I see lots of protocols using general primary splenocytes as feeder cultures (primarily due to the prevalence of DCs) but we're trying to avoid using animals for feeder cells and we figured a cell line may be good enough. Is that reasonable? But I'm assuming everything else (namely, irradiation) should be the same as for splenocytes? Would 5000 rad also be an appropriate dose?
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What is your goal? Just expansion of antigen specific CD8+ lines?
Even with the cells HLA matched the Cell line may present neoantigens to the CD8 T cells since the intracellular proteins of the immortalized cell might be different than of your mouse. That's not to say it won't work of course using immortalized cell lines is quite common, though typically autologous immortalized cells for antigen presenting cells.
The closer matched HLA the better inbred mice certainly have less HLA diversity than people so if the Mast Cell line came from the same strain it is more likely to work. As for other HLA antigens you can check one of the alloantigen lists: https://www.biolegend.com/Files/Images/media_assets/support_resource/BL_MouseAlloantigens_041116.pdf
The Controls you have seem fine I organized below for my understanding.
1. Mock Co-culture no TCR Stimulation: Controls for non-specific activation(HLA or other accessory cell effect)
2.Mock Co-Culture TCR stimulation: Postive control + Co-Culture control
3. Infected Mast: No T cell control(can be useful to confirm successful radiation and infection)
4. TCR Stimulation: Positive T Cell only control(shows cells can proliferate)
I think you can do without the Mock Co-Culture with TCR stimulation(2) if you had to drop one. I'm not sure what it is controlling for that your other controls aren't already covering. Depending on your methods and goals you could loose more of them ie you can loose control 3 if you can prove infection/successful radiation treatment in your co-cultures.
Could be interesting to see what happens with a immortalized Mast cell for co-culture. You would have to prove the T cell specificity to viral antigens of course. I think you would get cleaner data from autologous cells.
As for working with primary Immune cells they should be fine to leave overnight or 24 hours after isolation in complete media. Human T cells will sit there just fine for a little while, even longer if you give them Il-2 or some other survival factor that you will probably give them later anyway(might affect differentiation though!!). I'm not experienced with mouse T cells but I Imagine short term survivability isn't too different.
Furthermore depending on how long you have to infect your cells for you can incubate them with the virus for the necessary time for infection, wash them and then immediately go to co-culture while viral proteins start to be expressed.
I can envision a workflow where you homogenize Spleens, Isolate CD8(+) cells leave over night, Irradiate CD8(-), Infect overnight then mix the next morning. Alternatively use the previous mouse's CD8(-) Cells for the next mouse's CD8(+) expansion. Just freeze down the CD8(-) fraction when you get it(quickly as subpopulations will adhere to plastics and you will loose them) and then use those frozen cells infection and presentation to the next mouse's CD8(+) cells. That way you can prepare Infected cells the day before assuming freeze thaw doesn't affect infection rates.
No mater the method as long as you confirm the antigen specificity at the end of your experiment it may not matter how you got there.
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I have added anti- CD28/anti-CD3 beads to in vitro culture of murine T cells. Some of the cells have increased in size which indicates their activation. However, is there a method to quantify the activation of T cells? (I know IFN- gamma levels are measured but is it reliable?)
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Check out my paper on activation and survival of CD8 T cells. There you find protocols for most of the assays that Brett P Gross mentioned, including protocols for antibody/ CFSE staining and flow cytometry.
Please let me know, if you have any specific questions on how to set up the assays.
All the best,
Michael
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We are working with murine CD8 T cells at the moment and for this sake we isolate CD8 T cells with the Dynabeads untouched mouse t cells kit (thermo fisher). So for several weeks we didn't have any problems and a quite good efficency. But since three weeks we are struggling with the isolation because our cells cannot be stimulated and die after seeding them. We didn't change any steps in the protocol. So for better understanding this is the rough protocol we use:
- Smash the spleen with the end of a 2 ml syringe and filter throgh 40 um Nylon mesh
- suspend and wash the cells in cold PBS + 0,5% BSA + 2mM EDTA
- resuspend the pellet in 1x erylysis buffer for 2 min, fill up with cold PBS + 0,5% BSA + 2mM EDTA and wash
- block the cells with FCS and follow the instrustions from the isolation kit to isolate CD8 cells (we never changed how we did this: incubating cells with antibody mix, washing beads, incubate cells with beads, remove beads, transfer supernatant containing CD8 cells, wash beads)
- resuspend the isolated CD8 cells in media + 10% FCS, add 100 U/ml IL-2, add CD3/CD28 activator beads (Ratio 0,8:1), add 50 uM beta-ME
- seed the cells in a density of 1*10^6/ml in a 24 well plate
After 3 days we normally saw a good proliferation and cells sticking to the beads.
Since three weeks the cells just seem to die. After 3 days the density is no more than 0,2-0,3*10^6/ml. The cells don't stick to the beads and don't look stimulated. Using flowcytometry we sometimes saw a lot of celldebris (as we suspect) in our suspensions even shortly after the isolation.
We double checked every solution we used.
So we used fresh PBS +0,5% BSA +2mM EDTA and we diluted fresh 1x erylysis buffer from our 10x stock solution. Those solutions are being used by most of the people in our lab and no one esle is having any problems like this. We have two different operators doing the isolation. Each of them are preparing two different mice.
So we opened a new bottle of activator beads and saw no difference.
We then came to the conclusion that it might have something to do with the isolation kit. So we skipped the isolation in our protocol and stimulated the splenocytes which seemed to work as far as possible. We then used a different kit for a positive isolation and this worked too, but the efficency is quiet low which is why we would rather stick to the negative isolation.
We ordered another Dynabeads untouched mouse t cells kit with a different lot number which we used today.
We don't now yet if the cells can be stimulated but our flowcytometry analysis looked horrible. Again there was a lott of cell debris and only around 7% lymphocytes (normally around 80%).
So now my question is: does anybody have experience with a problem like this or any kind of suggestion what we could check again?
Thank you.
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Annick Fehrer Hi i read about your struggles, and I'm glad that you have solved the issue! I was, however, intrigued by your last message- do you have any sources where I could read about isoflurane impact on immune cells/ how did you pinpoint this as the problem? Thank you in advcance!
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Hello.
I work in a cancer immunology group. We need to deplete CD45 (.1 /.2) OT-1 cells previously transferred to CD45 (.1) mice, with aCD45.2 depletion antibody. But we have doubts about the dose to use. We usually deplete CD8+ T cells with 60ug of aCD8 depletion antibody (YTS169.4) but this time we need to deplete a smaller number of cells and maybe 60ug of aCD45.2 is an excess. In their work, Laura Mackay's team uses about 10ug of the antibody aThy1.1 (HIS57) to deplete CD8+ T cells Thy1.1+.
Beforehand thank you very much.
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I am so sorry for such a delay, I had not reviewed Researgate in many weeks.
Could you solve your problems ?. We have performed similar experiments and we have not had that problem, so I am not sure what it could be. One question, does your cell therapy promote tumor eradication?
I have seen that some researchers use treatments with IL-7 / IL-15 to favor memory phenotypes that last over time favoring the antitumor response.
It could also be that your B16 cells have lost the expression of OVA, do they have a probe to follow the expression of OVA ?.
I stay tuned in case you need help.
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I'm trying to understand a rather basic concept of T cell development and need some clarity. During self antigen recognition to generate tolerance, how or do cortical thymic endothelial cells present every possible self peptide that cells could generate (considering ~20000 protein coding genes). Is it possible that some peptides are not presented that form a self protein fragment and CD8 T cells mature without recognizing it as self. Now, can this self peptide that was missed during selection in thymus be presented at a later stage by a different cell to now be recognized by mature CD8s as non self? I could be totally wrong so apologies in advance.
Thanks
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We know that each person has many self-reactive CD4-T cells in their body, virtually everyone responds to myelin basic protein, for example. How do these cells escape deletion in the thymus? There are 3 papers describing mechanisms: first, the T cell epitope binds very weakly to MHC class II (Liu GY, Fairchild PJ, Smith RM, Prowle JR, Kioussis D, Wraith DC. Low avidity recognition of self-antigen by T cells permits escape from central tolerance. Immunity. 1995;3(4):407-415); second, the T cell epitope is not expressed in the thymus, in the very young, due to alternative splicing of the gene (Anderson AC, Nicholson LB, Legge KL, Turchin V, Zaghouani H, Kuchroo VK. High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire. J Exp Med. 2000;191(5):761-770) and third, the gene may be expressed but the protein is degraded by proteases in the thymus (Manoury B, Mazzeo D, Fugger L, et al. Destructive processing by asparagine endopeptidase limits presentation of a dominant T cell epitope in MBP. Nat Immunol. 2002;3(2):169-174). Cells escaping deletion in the thymus may be controlled by peripheral tolerance mechanisms including sequestration (being hidden away from the immune system), expression in a privileged site, homeostatic mechanisms and, of prime importance, immune regulation by regulatory T cells.
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I am trying to stimulate and expand t cells. I am using dynbeads to perform a negative selection for CD8+ cells from mouse spleen. after selecting CD8+ cells, I start of with around 1 million cells in culture (advanced rpmi with L-glut, sodium pyruvate, 10%FBS and Penstrep) with 10 ng/ml IL-2 and use Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation from thermo.
After culturing for 2 days I have a large decrease in viability and I am left with little cells.
I want to compare unstimulated and stimulated T cells in the T cell killing assay and have a few queries.
Can anyone recommend anything to increase the viability of cells? Do I need to culture the cells with the activating dynabeads for longer? Is this concentration of IL-2 sufficient - the protocol suggests 30U/ml - how can I convert this to a volume of IL-2?
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Actually, you should incubate the T cells with beads for a shorter period of time. Prolonged stimulus causes them to become anergic or undergo activation-induced cell death. I would limit co-culture with the beads to 24 hours maximum.
What kind of vessel are you culturing the cells in? After removing them from the stimulus, I find the cells tend to do best when you put them into U-bottom 96-well plates at an initial density of about 1 million cells/mL. So, in your case, if you're getting about a million cells after negative selection, you would briefly stim, remove the beads, adjust the cells to a volume of 1 mL, and split them across 5 wells of a U-bottom 96-well plate.
As for IL-2, be careful here. Every batch of IL-2 has its own activity as measured in bioactivity units (1 unit = the ED50 of metabolic activity in the cell line, CTLL-2). So there is not a single number you can use to convert between volume or mass and units. The best thing is to get the actual bioactivity assay data from your supplier OR perform your own in-house assay to determine activity (here's an example protocol, but there other variations too: ).
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Hello
I am having trouble getting clean proliferation signals in my CD8+ T cell assays. Generally, I stain with a CellTrace reagent (eg CellTrace Violet). When I stain naive T cells, activate (anti-CD3 @ 10 ug/mL, anti-CD28 @ 2 ug/mL, and IL-2 @ 25 U/mL) and then measure with a cytometer 2-3 days later, I get very clean signal that shows clear dilution of the stain. See attachment one (d3 001.fcs_Lymphocytes.png).
The problem arises when I restimulate T cells. The experimental design looks like this:
d0: activate (anti-CD3 @ 10 ug/mL, anti-CD28 @ 2 ug/mL, and IL-2 @ 25 U/mL)
d2: remove T cells from from anti-CD3 and anti-CD28, transfer to a new well with fresh media and 100 U/mL IL-2
d4: stain T cells with CellTrace, restimulate using a range of anti-CD3 concentrations (and 2 ug/mL anti-CD28), and seed in different media conditions that I am testing (I am interested in metabolism). IL-2 is lowered to 25 U/mL.
d6: measure proliferation using a cytometer
Please see the second attachment for staining results. Grey is unstained, red is a condition with little to no proliferation, and green is a condition with some proliferation.The problem is that I end up with these broad peaks, with no discernible cut-off points between divisions. I have been told that it is difficult to measure proliferation with restimulation because after the initial stimulation, all of the cells are cycling and not uniform in size, resulting in broad peaks. Does anybody have any suggestions?
My thought was to let the T cells rest off of stimulation for two days before staining and restimulating, perhaps to allow for a more uniform starting size at staining and restimulation, but it has clearly not worked. Perhaps resting the T cells with such a high concentration of IL-2 is contributing to the problem, promoting continual cell turnover?
Anti-CD3 is always plate bound, and anti-CD28 is soluble.
All thoughts and ideas are appreciated and welcome!
Matt
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Would it be possible to do cell trace in 2 different colors? Then you could plot cell trace stained d0 vs cell trace stained during restim. Maybe that would help with your spread in staining of stimulated cells.
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We are attempting to follow a Nature Protocol to separate activated T cells from unactivated T cells for retroviral transduction. ( )
Flow checks are showing no separation with Percoll gradients before and after despite following the protocol. Each layer (interface and bottom) has the same population percentages as the starting population. We do see activation for our murine CD4 T cells (~90%+ purity and 71% CD69 expression), though they are spread out across multiple populations in the SSC:FSC plot. Our CD4 T cells are better activated at 24h vs 48h.
Does anyone have experience with this protocol or something like it?
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Thank you Julie. We have already separated out CD4 T cells with an EasySep kit. Percoll, according to this protocol, is supposed to separate the larger blasting cells from the smaller inactive cells. We will contact the authors to see if they can give us specific advice for the protocol.
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We cultured antigen - specific CD8+ T cells for 3 passages with IL2 supplement (also stimulated once with CD3/CD28/CD2); we noticed a lot of elongated cells floating (not attached) within the culture (see the image attached - one view presented lots of those irregular - shaped cells). We have never seen these type of floating cells before by using the same parental T cells and the same culture condition.
Normally, after stimulating by CD3/CD28/CD2, we see lots of "spheres (clusters)" plus small round cells, not this type of elongated big floating cells. These cells are also grow super fast. It also seems that this CD8+ T cells lose its specificity by killing all type of cells regardless of antigen of HLA type.
Is it possible a contamination? or our culture agents are no longer good? Too much stimulation? Has anybody seen those big, elongated T cells in the culture? if they are activated CD8+ T cells, they should be round withing a cluster, instead of floating individually?
Thanks!
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You see normal long cultured CD8+ T cells. Usually they are shaped like worms. Big size and the loss of specificity may be due to too high concentration of IL-2.
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HI! I am working on a project invovling CD8+ T cell culture in vitro.And the cd8+ t cell are isolated directly from mice spleen.i am wondering after i activated cd8+ t cell with CD3 and CD28 antibody at both 1ug/ml and IL-2 at 100U/ml in 1640 medium ,should i remove the cd3 and cd28 from the medium,in the same time,keep il-2 in the medium?Thanks!
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No, you should not to do this. You can find concentrations of antibodies, which will provide stable growth of CD8 T cells. Concentration of IL-2 seems too high - you have risk to induce formation of LAK-cells, which will kill your cultures.. 5-10 U/ml would be better.
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Hello,
I want to stimulate my splenic mice cells in vivo to let them expand and proliferate more before I isolate them. Does anyone knows a in vivo method to do so?
thank you!
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Dear Natallia, poly I:C is well known ligand for TLR3 on DC and macrophages. Could you explain the mechanism of activation CD8 T cells by this substance?
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Hi, guys
I`m quite fresh in the field of culturing CD8 T-cells in vitro and I need an advice. I want to test if a population of CD8 T-cells may give protection against LCMV-Armstrong virus infection in mice. So, we want to isolate the cells from LCMV-immune mice, expand, then transfer into naive hosts and challenge the mice with LCMV. As a measure of protection we want to use viral titer. We rather doubt if the cells are protective.
The point is that antigen specificity of the overall cell population is not known. The cells of interest contain some antiviral effectors, but relatively few and it`s not known whether these effectors play any role in the response.
TCR-transgenic mice are unavailable as far as induction of the marker I`m studying is not possible to achieve.
So, I`d avoid antigen-mediated CD8 T-cell expansion as far as it can promote some cell while depleting the rest from the population of interest. Initially I planned to use IL15-mediated expansion. Preliminary tests with that cells from naive mice were promising for 50 and 100 ng/ml of IL15. Cells grew very fast and within 1 week I was able to get 5-6 millions cells starting from 0.1 million of sorted cells.
I am making a preliminary trial with total memory CD8 T-cells from LCMV-immune mice. The cells grew quite fast on IL15 and expanded more than 20 times within 1 week. They were viable, proliferated in response to LCMV-peptides and secreted IFNg.
I wanted to expand them more to make some additional tests, but after 1 week of culture in vitro the cells stoped growing. Right now 1 week passed, but they hasn`t expanded even a little. They are just alive, neither die, nor proliferate.
Unfortunately, after stimulation with plate-bound aCD3e (1 mcg/ml overnight) that IL15-expanded CD8 T-cells from LCMV-immune mice died very fast. So I`m not sure if I can use this method.
Any ideas how to expand them? Should I try weak aCD3 stimulation with plate-bound antibodies and cytokines? Or I`d rather try some stimulator cells with soluble aCD3? Or some cytokines? IL15/IL15Ra?
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Correct...
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I plan to isolate and culture CD* T cells from MS patients and further transfect them and study the changes. How can I maintain them in vitro without further stimulation?
Can I go on with complete RPMI media only without IL-2 or anti-CD3?
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Hi Aya,
I've kept primary T cells alive for quite a while (CD8s) by a regular regimen of rest (low IL-2) and brief expansion (high IL-2 + CD3/CD28/CD2--beads are fabulous--stemcell has some lovely ones). IL-7 is also helpful.
T cells will go away if they think they're not needed, so periodic stim is important...
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i started a project invovling cd8 t cell in vitro expansion,recentlly.i isolated the cd8 t cells from mice spleen and started to culture the cells at 1*106/ml.But i don't know if i can remove the cells into new wells,when the numer of cells increase to a certain number .
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In general, there is no problem for two weeks at the concentration of 1x105.
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I am working with CD8 T cells +/- aCD3/aCD28 and I am trying to perform a WB from lysate from these cells. I optimized all the antibodies and protocols on PBMC +/- aCD3/aCD28 cell lysate, but when I had to do the actual experiment with my T cell samples because of reasons outside of my control (i.e. faulty power source during transfer) I was forced to freeze/thaw my samples several times (about 5 times).
I am storing my samples in Rippa buffer (50mM Tris pH 8, 150mM NaCl, 0.5% DOC, 1% NP-40, 0.1% SDS) at -80 degrees. From each sample I have something like 30ul with about 120ug of total protein.
Does anybody have experience in working with CD8 T cells?
Is the way I store my cell lysate unappropriated?
Any suggestion?
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Repeated freezing and thawing might shere the protein and it appears as a smear in sds- page.