- Jie Deng added an answer:3Can anyone help me with T cell differentiation analysis with anti-cd3/cd28 stimulation?
I am trying to observe differentiation of T cells by using different type of ligands( PolyI:C, CpG ODN,R848). Until now, I coated my plates with Anti-cd3 antibodies ( 5 ug/ml) and gave soluble anti-cd28 (1 ug/ml). T cells stayed in coated plates for 3 days in the same medium( 10%RPMI w/ 0.5mM ME) and checked IFNg, IL2 secretion by ELISA. However, all my groups' IFNg responses were so high, I could not differentiate the concentration difference between groups. Also, I tried to stain my T cells with anti-CXCR5 antibodies to see Th1 differentiation. However, cells were getting smaller and becoming unhealthy. I could not get any clear results.
Can anyone help me to understand what I am doing wrong?
Thank you in advance;
- Benjamin G Vincent added an answer:4Can T cell receptor sequencing be used to simultaneously quantify and characterize various T cell subsets after cell culture?
I would like to characterize changes in the T cell receptor repertoire after ex vivo stimulation with different types of antigens (conventional peptide/MHC, unconventional antigens, superantigens, other unspecific proliferation inductors). Specifically, I would like to simultaneously quantify expansion of T cell types (gamma/delta vs alpha/beta), certain chains (like changes in Vbeta-profiles after SAg stimulation), T cells with (semi-)invariant TCR chains (like Vg9/d2 cells) and single clones (CDR3-sequences), in order to screen unconventional T cell antigens for their effects.
I am very unsure of how to approach this. I can find quite a lot of information on CDR3 sequencing, but most papers either focus only on specific T cell subsets or try to determine the overall TCR diversity in an individual. I still don't know, whether information from CDR3 is sufficient to determine which genes the respective cell used when creating the TCR. Can the CDR3 sequence be linked back to the corresponding alpha, beta, gamma or delta chains or do I need to analyze different regions in order to see, for instance, an expansion of Vbeta2?
Are there universal primers to capture the whole TCR-repertoire within my culture? Currently I intend to rewrite and expand the TCR-RNA in an RT-PCR. How can I ensure that the semi-quantitative nature of RT-PCR is sustained when primers bind to different chains with different affinity? Is there a bias?
I hope you can give me some pointers or "must-reads" for TCR sequencing.
I really appreciate any help you can provide.
Thanks Johannes. This is a nice method - Getting the RNA digestion right seems to be key. I haven't tried this - We are pursuing the RACE method with longer-read sequencing for now.Following
- Ekaterina Victorovna Moiseeva added an answer:5I want to do rat spleen lymphocyte culture, do I need to keep spleen on ice?
I'm going to culture spleen lymphocyte from rat by following step
1. remove spleen
2. soaking with 1X pbs (about 6 hr. before culture)
3. Mince rat spleen mix with RPMI+10% FBS and pass through sterile gauzze
4. lysis RBC with RBC lysing solution
5. Centrifuge at 2000 rpm and culture on plate
***I want to know that which step I could keep on ice or any protocol suggest me?
I wish you good luck!Following
- Fana Alem Kidane added an answer:4Is there any chance of rescuing protein targets in thawed and re-frozen cryo-tissue samples ?
The samples were liver, spleen and others intended for immunohistochemical staining of CD8 and CD4 T cells. They were initially snap frozen with cryoprotectant (OCT) in liquid nitrogen. I found them thawed due to a freezer problem. I snap froze them again anyway to just bring their temperature back to freezing and see if there is any chance of rescuing some targets if not all. Has any one had such an experience or any thoughts how this should be addressed? Thank you very much.
Yea, I also think so. Thank you.Following
- Vineeth Varanasi added an answer:9How can I get rid of peripheral CD4 T cells in BALB/C mice at a certain point of time in their life without using antibodies?
I want to reconstitute the mice with genetically modified CD4 cells after withdrawal of endogenous CD4 cells. Thus, since antibodies are quite stable, I guess antibody-mediated depletion is not the method of choice. Any suggestions?
You can get reversible (but may not be 100%) depletion of T cells using dexamethasone treatment. Here's a link to a paper on that:
Maximiliano's wish has already come true. Jackson labs does sell a mouse with cre inducible DTR mouse on the B6 back ground ( Stock No:007900 | ROSA26iDTR ). Depletion is much better in this mouse, although titrating the exact diptheria toxin dose to achieve good depletion can be quite a pain and is complicated by the toxicity of diptheria toxin.
- Durjoy Majumder added an answer:5How can I isolate lymphoid and myeloid leukemic cell from Ethylnitrosourea induced mixed type leukemia?
In BALB/c mice treatment of Ethylnitrosourea can produce leukemia but both lymphoblast and myeloblast cells are present. How Can I isolate only lymphoblast or only myeloblast cells from the bone marrow or from the peripheral blood?
For human leukemia: NO - aberration means no specificity; for ENU induced leukemia: you have to find out.Following
- Florian Ebner added an answer:8Any issue with the staining of T panel surface markers after fix&perm protocol?
Hi, I am combining intracellular staining of phosphoproteins with common surface markers (CD3, CD4, CD8, CD25, CD62L, CD44), in mouse splenocytes for flow cytometric analysis. I first do the surface staining, then stimulate the cells, then fix & perm with the saponin or mild alcohol method , and then finally the intracellular staining, and all works well. But for my purposes I'd rather prefer combine the surface and intracellular staining after the stimulation and fixation period. It is described any weird thing with the staining of these surface markers after the fixation and permeabilization with saponin or alcohol (in terms of problems with antibody binding, etc? Thanks in advance for your advice.
Out of lazyness, I fixed already often splenocytes and combined intrazellular and extrazellular stainings ... from the markes you mentioned, only CD62L was never working for me - the signal was just gone. But as mentioned before, that might be a clone-depenend thing too ... not only matter of expression, as I used unstimulated mouse splenocytes ex vivo ...Following
- Olivia B Harris added an answer:1Does anyone have a protocol for isolation of TILs from B16F10 tumor using percoll or Ficol?
My aim is to isolate TILs from B16F10 tumor. I used Discontinuous method using 80% and 40 % percoll.
I get a concentrated tumor cells above 40% percoll as shown in picture.
The tumor cells are very sticky, and stick to tips when I try to remove them.
Has any one tried isolating TIL from B16F10 tumor?
I have isolated TILs from other syngeneic tumours but I know that B16F10 tumours can be a tricky tumour to work with. I have used the miltenyi gentleMACS kit and that worked well but I know it can be very expensive. You could try coating your tips with EDTA before removing the cells, then washing with PBS and FCS / EDTA? Hope this helps, good luck.Following
- Simon Gebremeskel added an answer:3How can I detect Treg cells from peripheral blood?
I want to detect Treg cells from peripheral blood, and I can use PBMC, but when I try to do directly the whole blood I got CD4+CD25+Foxp3+Treg cells and I found similar result with previously reported % of Treg cell using PBMC; do you think my experiment is wrong?
I use BD Transcription Factor Buffer Set. Material Number 562574, and BD FACSCANTO II.
I think they approach yo are taking is correct. Just depends with how you represent the data and the gating strategy you use.Following
- Per H Larsson added an answer:1What are the immune cell's composition (in percent) in sheep PBMCs?
Mainly looking for CD14, CD4, CD8, gamma-delta T-cells, NK cells and B-cells.
Have you seen this?
- Ketil Winther Pedersen added an answer:5Is there an antibody available commercially for expansion and selective enrichment of human CD4 or CD8 T cells in culture?
I want to generate CD4 or CD8 T cells from PBMC in culture with minimal manipulation (i.e. no antigen stim and no flow/magnet sorting). Years ago I used a CD3,4b bi-specific monoclonal antibody from J. Wong (Jones et al J Immunol Meth 274:139, 2003) that acted by negative selection to enrich for CD8 T cells, and vice versa for CD3,8 mAb and CD4 T cells, both of which worked really well. Now I can't find similar reagents available commercially...
I have used Dynabeads CD3/CD28 to expand T cells, but I am looking for CD4 or CD8 enrichment specifically. Thanks for any suggestions on finding these reagents.
Hi, for isolation of CD4+ or CD8 positive cells from PBMC an alternative is to use the Dynabeads Untouched CD4 T-cell Kit/Dynabeads Untouched CD8 T-cell kit. Both kits are compatible with downstream activation or expansion using Dynabeads CD3/CD28 beads. Feel free to contact me at any time. kind regards, ketilFollowing
- Raul Jimenez Heredia added an answer:3Which is the best way to isolate lymphocytes from a renal biopsy?
I am currently working on characterization of lymphocytes extracted from diseased kidney tissue (i.e. acute rejection). Until now the piece of kidney that we received was big enough to extract lymphocytes just by mechanical dissection, but in the next weeks we will receive small biopsies and I am not sure if mechanical dissection will be enough. Also, I have read that with enzymatic digestion the surface markers can be disrupted by the collagenase (and I need them for further FACS analysis). Any suggestion?
Thank you in advance!
Thank you very much for your answers. For the first samples we will try mechanical dissociation manually and then probably collagenase IV, that has very low concentration of trypsin. If it works out we will think about the Medimachine.
Ishak, can I ask you if you have used before the Medimachine for lymphocyte isolation from tissue or if you know any specific publication related?
Thank you in advance, and I will describe the results after the first try.Following
- Dirk Werling added an answer:2What is the procedure to isolate mononuclear cells from milk of buffaloes for FACS analysis ?
I want to analyse CD4 and CD8 cells from milk by Flow cytometry.
Mahavir, unless your buffaloes have mastitis, you won't find hardly any Tc in normal milk. The majority of cells will be epi-/endothelial cells, macrophages and granulocytes. Very tedious to sort T cells from milk. In some cases, we started of with a couple of litres to obtain 10e7 T cells. Remember, normal lymphozyte migration into the udder doesnt happen until blood-udder barrier is disturbed.Following
- Douglas G Mack added an answer:9How can I make the Mouse Treg Suppression Assay work?
I would like to investigate the suppressive capacity of Tregs isolated from mouse spleens and lymph nodes. I already did the experiment twice but in both cases the responder T-cells proliferated MORE when Tregs were present.
We FACS sorted CD4+, CD45RBhigh, CD25- as naive responder T-cells and the Tregs as CD4+ CD45RBlow, CD25+. The stimulus was plate-bound CD3/CD28 and in a second approach we tried a low PMA/Iono concentration. However, co-incubation of 100 000 naive T-cells together with 100 000 Treg lead to enhanced proliferation.
So I thought that in the population we sorted as "Tregs" there may be some activated T-cells present. That's why we included CD62L for sorting of the Tregs in our second experiment. The stimulus was again plate-bound CD3/CD28 and this time we also tried CD3/CD28-Dynabeads in parallel. But the problem of enhanced proliferation remains.
Does anyone have experience with mouse Treg suppression assays and can give some advice?
A few good points in the above responses.
Sort on Cd25hi for Treg (test with FOXP3 intranuclear staining)
Try irradiated CD2- cells as APC's (no anti-CD28) and titrate the amount of anti-CD3.
Detection: Might consider labeling your effectors with a dye, like CellTrace VioletˇFollowing
- Ayşe Balamir added an answer:2Does anyone know about a human oligodendrocyte with naive human CD4 T cell co culture method?
I need some information indirect co culture or firstly about primary CD4 T cell culture method. I am planning PBMC from human blood then I am using human CD4 T cell isolation kit II (MACS) for CD4 T cell culture. But I don't know necessary reagent for first primary culture and after needed for co culture with commercial human precursor oligodendrocyte?
Thank you, Daniel
actually I want to know Th17 cells isolation from PBMC and after planning to use naive human CD4 T cell isolation Kit II for CD4 T cell culture with specific reagents and following FACS but I needed some information or protocol for using reagents that I haven't know yet and visualizing Th17 cells on FACS.Following
- Mehdi Mahdavi added an answer:3Which antibody manufacturer should I choose?I don't have much experience and I need to analyze some cell markers on flow cytometry as CD38 on human lymphocytes. Does anybody know if Biolegend antibodies are a good choice? Or which manufacturer should I choose?
RnD system , BD-Bioscience, eBioscience........Following
- Mehdi Mahdavi added an answer:7Does anybody have experience in and/or a protocol for the isolation of lymphocytes from gut tissue?
I'm struggeling to get lymphocytes out of gut tissue (inflamed small intestine). We have been using different percoll gradients and protocols, but nothing worked out. Would be great if anyone could help! Thanks a lot!!
Separate gut tissue and wash with PBS. Then dissect and then digest with Collagenase 1mg/ml, DNAse 50 mic/ml, Hyaloronidase for 45 min at 37 degree C and after pipeting the sample should pass through cell 100 mic mesh and lymphocyte seperated using ficole method.Following
- Anne Lodge added an answer:4Which is the best of the different sources for mouse CD4+ T cells, spleen or lymph?
Can anyone explain, which source is best for isolation of mouse CD4+ T cells for basic functional assays, spleen or lymph nodes?
In addition, I haven't noticed anyone isolating CD4+ T cells from mouse peripheral blood. Is there a particular reason for this?
I've tried to get mouse PBMC and it is just not easy. It seems like there are more rbc and it is harder to get separation of the mononuclear cells from the rbc. And remember that from a single mouse you can get perhaps 1 mL of whole blood but from a human it is easy to get 50 mL. The lymph nodes and spleens are just packed with lymphocytes. If we could easily get those from humans we'd be doing that instead of blood.Following
- Anh Do added an answer:13Do you know the easiest way to isolate lymphocytes in lamina propria in an experimental mouse model?I'm doing an experiment about inflammatory bowel disease in mouse models. I performed IHC in murine colonic tissue, and the lamina propria lymphocytes were well stained with the primary antibody. Next, I want to estimate the mRNA and protein levels in lamina propria lymphocytes.
I know there are several ways to isolate lamina propria lymphocytes in mouse models, However, they are very complex and time-consuming. These ways also require much trial and error. In addition, I don't have the reagents necessary for the isolation methods. Do I have to buy all the reagents, or is there another way to source them?
I would like to know the easiest way to isolate LP lymphocytes in mouse model.
Hi Ekaterina Vladimirovna Sidorova, Jon Jo
How do you dissolve 5mM EDTA? And what is the final pH of the EDTA solution used to disrupt epithelial cells?
Thank you very much.Following
- Sergei B Koralov added an answer:7Do you have suggestions for a good method to extract lymphocytes from mouse skin for cytokine profile analysis?We are looking to extract CD4+ T cells from diseased mouse skin for lymphocyte analysis. So far the protocols we have tried either yield only ~500 cells from a 1 cm sq of skin (healthy c57Bl/6 mice) or have such a harsh liberase treatment that some markers such as CD4/8 that we need for our FACS analysis are lost and all are rather inconsistent. If anyone has a good protocol we would really appreciate your advice!Hi Prem,
Thanks for the link. Indeed we've considered Dr. Clark's and Dr. Kupper's method and there are certainly advantages to it, I am a bit hesitant to adapt it for our purposes because the extravasation of T cells into the 3D matrices is accompanied by >10x expansion and by significant molecular changes in the cells including upregulation of the activation markers. While the clonality of the cells should not change, I am concerned that the nature of the cells may be quite different - as we are analyzing tumor cells and are also transferring the T lymphocytes into RAG KO and B6 hosts, I am a bit concerned about changes that may take in these cells in the course of extravasation...Following
- Melanie Dimapasoc asked a question:OpenHas anyone used SepMate tubes + Ficoll for PBMC isolation?If so, have you noticed any difference in RBC, monocyte and/or granulocyte contamination, compared to using Ficoll alone? Also, have you noticed any difference in lymphocyte yield between the two procedures? I am running some validation studies to compare the two separation procedures and have been having some issues with contamination and decreased lymphocyte yields over time using SepMate + Ficoll.Following
- Marina Ninkov asked a question:OpenCan anyone help me with lymphocyte isolation from a rat's small intestine?Is it possible to use 2-mercaptoethanol instead of DTT?Following