کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8504708 | 1554986 | 2018 | 34 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood
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کلمات کلیدی
FACSqRT-PCRCD8CD4MACSMin - حداقلminutes - دقایقB cells - سلول های BT cells - سلول های تیfluorescence activated cell sorting - فلورسانس سلول فعال فعال سلولmagnetic activated cell sorting - مرتب سازی سلول های فعال مغناطیسیquantitative reverse transcription polymerase chain reaction - واکنش زنجیره ای پلی مراز رونویسی معکوس
موضوعات مرتبط
علوم زیستی و بیوفناوری
علوم کشاورزی و بیولوژیک
علوم دامی و جانورشناسی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Immune phenotyping of equine peripheral blood mononuclear cells (PBMC) is commonly described by single or double marker labeling, which limits complex phenotypic descriptions and subpopulation identification. Our objective was to develop a new multispectral flow cytometry protocol to identify and sort equine lymphocyte subpopulations using commercially available, pre-conjugated monoclonal antibodies to cell surface markers. Two clones of anti-equine CD8 (CVS8 and CVS21) were compared in combination with CD3. Clone CVS21 bound non-T CD3â cells in addition to CD8+ T cells. Further analysis using co-labeling with CD3 and multiple B cell antibodies revealed that most of these CVS21+CD3â cells were B cells. Clone CVS8 and anti-equine CD4 clone CVS4 only labeled CD3+ cells and were mutually exclusive. To identify equine B-cell subpopulations, anti-equine Pan-Ig (clone CVS36), IgM (clone 1-22), and IgG1 (clone CVS45), as well as anti-human CD21 (clone B-ly4) were tested. Anti-equine Pan-Ig antibody labeled 88â¯Â±â¯7.6% of CD3â lymphocytes. Anti-equine IgM often produced a continuum of stain intensity of PBMC from several adult horses. Anti- IgG1 and âCD21 labeled overlapping subsets of CD3â cells. Based on these results, combined with conjugate availability, a final panel of anti-CD4 (CVS4), âCD8 (CVS8), âIgG1 (CVS45), and âCD21 (B-ly4) antibodies was used to sort CD4+ T cells, CD8+ T cells, and CD21+ and/or IgG1+ B cells simultaneously. The identity of the sorted populations was confirmed by qRT-PCR for cell lineage markers. The described analysis emphasizes the need for increased availability of reagents and use of multispectral flow for equine immunophenotypic analysis. This new multispectral flow cytometry protocol will facilitate studies on equine lymphocyte responses and will have broad application across studies of infectious and immune-mediated disease.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Veterinary Immunology and Immunopathology - Volume 199, May 2018, Pages 22-31
Journal: Veterinary Immunology and Immunopathology - Volume 199, May 2018, Pages 22-31
نویسندگان
Joy E. Tomlinson, Bettina Wagner, M. Julia B. Felippe, Gerlinde R. Van de Walle,