Intracellular Flow Cytometry Staining Protocol: For the Detection of Intracellular Cytokines and Other Intracellular Targets v1

protocols.io ◽  
2016 ◽  
Author(s):  
BioLegend not provided ◽  
Inc not provided
2017 ◽  
Vol 47 (8) ◽  
pp. 1389-1392 ◽  
Author(s):  
Katharina Pracht ◽  
Julia Meinzinger ◽  
Patrick Daum ◽  
Sebastian R. Schulz ◽  
Dorothea Reimer ◽  
...  

protocols.io ◽  
2016 ◽  
Author(s):  
BioLegend not provided ◽  
Inc not provided

2022 ◽  
Author(s):  
Patrick Renner ◽  
Michale Crone ◽  
Matthew Kornas ◽  
KimAnh Trang Pioli ◽  
Peter Dion Pioli

Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that antibody-secreting cells regulate multiple aspects of biology through the secretion of various cytokines. In this regard, intracellular flow cytometry is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Here, we showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of antibody-secreting cell intracellular protein expression compared to other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within antibody-secreting cells. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between antibody-secreting cells and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen antibody-secreting cells expressed Toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β.


Rheumatology ◽  
2020 ◽  
Vol 59 (11) ◽  
pp. 3340-3349
Author(s):  
Chien-Sheng Wu ◽  
I-Tsu Chyuan ◽  
Yen-Ling Chiu ◽  
Wen-Lin Chen ◽  
Chieh-Yu Shen ◽  
...  

Abstract Objectives SLE is an autoimmune disease characterized by aberrant autoantibody production and immune dysfunctions. Whether the anti-CMV immunity is impaired in SLE patients is poorly understood. We investigated the specific anti-viral T-cell response in SLE patients with CMV infection and its possible impacts on clinical manifestations in lupus. Methods CD28 null T-cell percentages were measured by flow cytometry in 89 SLE patients and 58 healthy controls. A specific anti-CMV CD8 T-cell response was assessed ex vivo by the production of intracellular cytokines in response to CMV phosphoprotein 65 (pp65) by flow cytometry. Clinical manifestations and immune parameters were analysed in SLE patients according to their CMV serostatus. Results CD28 null T cells were significantly expanded in SLE patients. When the anti-CMV pp65 CD8 polyfunctional T cell response was analysed, as defined by production of at least three of four functional cytokines or effectors (intracellular IFN-γ, IL-2, TNF-α and surface CD107a), the results demonstrated that it was not impaired in SLE patients. In contrast, when comparing clinical manifestations, there were lower anti-ds-DNA levels and decreased SLEDAI in SLE patients with CMV infection. Furthermore, the expansion of CD4+CD28 null T cells was negatively associated with anti-ds-DNA levels and SLEDAI in these lupus patients. Conclusion In SLE patients with CMV infection, the specific anti-CMV CD8 T-cell response is preserved but is associated with decreased disease activity and lower anti-DNA levels among these patients, suggesting CMV infection may mitigate lupus activity.


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