scholarly journals Computational Analysis of Multiparametric Flow Cytometric Data to Dissect B Cell Subsets in Vaccine Studies

2019 ◽  
Vol 97 (3) ◽  
pp. 259-267 ◽  
Author(s):  
Simone Lucchesi ◽  
Emanuele Nolfi ◽  
Elena Pettini ◽  
Gabiria Pastore ◽  
Fabio Fiorino ◽  
...  
Keyword(s):  
B Cell ◽  
Computational Analysis ◽  
Flow Cytometric Data ◽  
Flow Cytometric ◽  
B Cell Subsets

scholarly journals Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2633
Author(s):  
Casper Marsman ◽  
Tineke Jorritsma ◽  
Anja ten Brinke ◽  
S. Marieke van Ham
Keyword(s):  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Intracellular Signaling ◽  
Simultaneous Detection ◽  
Signaling Proteins ◽  
B Cell Differentiation ◽  
Flow Cytometric ◽  
B Cell Subsets ◽  
Human B Cell

The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. This will aid the further investigation of B cell responses in both health and disease.

scholarly journals Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

2020 ◽  
Author(s):  
Casper Marsman ◽  
Tineke Jorritsma ◽  
Anja ten Brinke ◽  
Marieke van Ham
Keyword(s):  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Intracellular Signaling ◽  
Simultaneous Detection ◽  
Signaling Proteins ◽  
B Cell Differentiation ◽  
Flow Cytometric ◽  
B Cell Subsets ◽  
Human B Cell

Flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help elucidate the regulation of B cell survival, proliferation and differentiation. However, simultaneous detection of signaling proteins or TFs, with membrane markers (MM) can be challenging as required fixation and permeabilization procedures can affect functionality of conjugated antibodies. Here, a phosphoflow method is presented for detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6 together with B cell differentiation MM CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows detection of B cell TFs; PAX5, c-MYC, BCL6, AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s together with MM. Applying these methods on in vitro induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and UMAP analysis revealed heterogeneity in TF-expression within stimulated CD27 or CD38-expressing B cell subsets. The methods presented here allow for sensitive analysis of STAT and NF-κB p65 signaling and TFs together with B cell differentiation MM at single-cell resolution. This will aid further investigation of B cell responses in both health and disease.

CD43 and CD5 antibodies define four normal and neoplastic B-cell subsets: A three-color flow cytometric study

Cytometry ◽  
1995 ◽  
Vol 22 (3) ◽  
pp. 223-231 ◽  
Author(s):  
Edward F. Lynch ◽  
Patricia A. Jones ◽  
Steven H. Swerdlow
Keyword(s):  
B Cell ◽  
Color Flow ◽  
Flow Cytometric ◽  
Flow Cytometric Study ◽  
B Cell Subsets

Lymphoproliferative disorders (LPD) involving lungs: T and B cell subsets within pulmonary infiltrates and in peripheral blood

1984 ◽  
Vol 42 ◽  
pp. 700-701
Author(s):  
Irene Stachura ◽  
Milton H. Dalbow ◽  
Michael J. Niemiec ◽  
Matias Pardo ◽  
Gurmukh Singh ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoproliferative Disorders ◽  
Mixed Population ◽  
Lymphoid Cells ◽  
Lymphomatoid Granulomatosis ◽  
Cell Type ◽  
Cell Surface Antigens ◽  
Pulmonary Infiltrates ◽  
B Cell Subsets

Lymphoid cells were analyzed within pulmonary infiltrates of six patients with lymphoproliferative disorders involving lungs by immunofluorescence and immunoperoxidase techniques utilizing monoclonal antibodies to cell surface antigens T11 (total T), T4 (inducer/helper T), T8 (cytotoxic/suppressor T) and B1 (B cells) and the antisera against heavy (G,A,M) and light (kappa, lambda) immunoglobulin chains. Three patients had pseudolymphoma, two patients had lymphoma and one patient had lymphomatoid granulomatosis.A mixed population of cells was present in tissue infiltrates from the three patients with pseudolymphoma, IgM-kappa producing cells constituted the main B cell type in one patient. In two patients with lymphoma pattern the infiltrates were composed exclusively of T4+ cells and IgG-lambda B cells predominated slightly in the patient with lymphomatoid granulomatosis.

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