Lymphocyte Subset Isolation

Lymphocyte Subset Isolation

  • Ayşe Balamir asked a question:
    Does 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?

  • Laura Gibson added an answer:
    Is 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.

    Laura Gibson · University of Massachusetts Medical School

    Hi Anna,

    Thanks for the feedback.  We are studying virus (CMV, HHV6)-specific T cell responses in young infants, so we have very few cells to work with...hence the approach of generating more T cells in vitro. We have used CD3/CD28 stim, but the CD4 and CD8 T cells tend to compete with each other leaving the the yield suboptimal for both.  The previous CD3,4b mAb worked really well for generating CD8 T cells...but that reagent is gone and I can't find any similar.  Might end up using negative selection beads, but the problem is cell loss during in the process, which seems to outweigh the benefit.  I would welcome any other suggestions..thanks again!

  • Mehdi Mahdavi added an answer:
    Which 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?
    Mehdi Mahdavi · Institut Pasteur International Network


    RnD system , BD-Bioscience, eBioscience........

  • Mehdi Mahdavi added an answer:
    Does 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!!

    Mehdi Mahdavi · Institut Pasteur International Network


    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.

  • Anne Lodge added an answer:
    Which 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?

    Anne Lodge · Astarte Biologics, LLC

    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. 

  • Anh Do added an answer:
    Do 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.
    Anh Do · University of Queensland

    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.

  • Sergei B Koralov added an answer:
    Do 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!
    Sergei B Koralov · NYU Langone Medical Center
    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...
  • Melanie Dimapasoc asked a question:
    Has 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.
  • Marina Ninkov asked a question:
    Can anyone help me with lymphocyte isolation from a rat's small intestine?
    Is it possible to use 2-mercaptoethanol instead of DTT?

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