The phenotyping was performed in whole blood within 4 hr. Absolute cell count was measured using the BDMultitest™ 6-color TBNK reagent with BD Trucount™tubes (BD Biosciences, Heidelberg, Germany). Employing 4 phenotyping panels, a comprehensive analysis of peripheral blood leukocytes was performed. The panels are modified recommendations of the “Human Immunophenotyping consortium” [16]. Briefly, Panel-1 consisted of 17 antibodies to characterize the major T-cell populations, including naïve and various memory/effector populations (CD2; CD3; CD4; CD8; TCR ƴδ; CD45RA; CD197 (CCR7); CD28; CD57 and CD127). Th1, Th2; Th17 and Tfh cells were identified with the following antibodies: CD183 (CXCR3); CD185 (CXCR5) and CD196 (CCR6). Activation was monitored with antibodies against HLA-DR, CD38, CD278 (ICOS) and CD279 (PD1). Using CD3 as a backbone for absolute quantification, with this comprehensive panel more than 200 defined subpopulations have been quantified in absolute numbers as well as in various ratios. Panel-2 consists of 8 parameters (CD3; CD19; CD20; anti-IgD; CD27; CD10; CD24 and CD38), allowing the identification of transitional, naïve and memory B-cells as well as circulating plasma blasts both in absolute numbers (CD19 backbone) and in various ratios. Panel-3 identified various NK, monocyte and DC subsets and included antibodies with the following specificities: CD11c; CD123; CD14; CD16; CD19; CD2; CD20; CD3; CD45; CD56; CD57; CD8a; HLA-DR; NKG2C and M-DC8. Using NK-cells as backbone, absolute numbers were calculated for 50 different populations. Finally, the Treg subset was characterized using the following antibodies: CD127; CD194 (CCR4); CD25; CD3; CD4; CD45; CD45RO and anti-HLA-DR. CD4+ T-lymphocytes served as backbone for the absolute quantification of the different subsets. The analysis was performed on an LSR Fortessa Analyzer (Special Order Research Product) (BD Biosciences).The detailed setup, staining, gating and analysis procedures have been recently described [17].

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.