Nurse-like cells or Peripheral blood mononuclear cells isolated from CLL patients or HD were treated with ibrutinib or acalabrutinib for 24 h and stimulated with germinated boiled killed A. fumigatus inactivated conidia or zymosan and analyzed using cytokine secretion assay (CSA) for TNF-α according to manufacturer’s instructions (CSA Detection kit; Miltenyi Biotec). Cells were immunostained with TNF-α catch reagent and incubated for 2 h at 37°C to allow cytokine secretion. After washes, cells were labeled with TNF-α Detection antibody conjugated to PE. To identify the monocytic population, PBMCs were stained with CD14 APC antibody. An isotype control sample for each condition was acquired to exclude autofluorescence background.
Csa detection kit
Manufactured by Miltenyi Biotec
Sourced in Italy
The CSA Detection kit is a lab equipment product designed to detect and analyze cell surface antigens (CSA). It provides a reliable and efficient method for the identification and quantification of specific cell surface markers. The kit includes the necessary reagents and protocols to facilitate the detection process, enabling researchers to gather critical data about cell phenotypes and populations.
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4 protocols using csa detection kit
1
Ibrutinib and Acalabrutinib Modulate Antifungal Immunity
2
Quantifying IL-10 Secretion in NLCs
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To determine IL-10 secretion, NLCs were cultured for 10 days and then treated with ibrutinib for 24 hours and analyzed using CSA for IL-10 according to manufacturer's instructions (CSA Detection kit; Miltenyi Biotec). NLCs were immunostained with IL-10 catch reagent and incubated for 2 hours at 37°C to allow cytokine secretion. After washes, cells were labeled with IL-10 Detection antibody conjugated to PE and CD14 APC Ab. An isotype control sample for each condition was acquired to exclude autofluorescence background.
3
Characterization of MM-specific T Cells
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A flow cytometry-based, phenotypic, and functional characterization of IFNγ-producing MM-specific T cells was carried out in two selected patients with robust responses in the ELISpot assay, by applying the CSA Detection Kit (Miltenyi Biotec, Bologna, Italy), according to the manufacturer’s instructions and protocols we previously described [34 (link),35 (link)], using, as specific antigenic stimulation, the 10 MMAA-derived peptide pools, individually. T-cell phenotype and memory profile of cytokine-producing lymphocytes were assessed after sample counterstaining with monoclonal antibodies for flow cytometry, including: CD3, CD8, CD4, CD62L, and CCR7 (Miltenyi Biotec, Bologna, Italy).
4
Cytokine Profiling of NPM1-Mutated T Cells
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The functional and phenotypic characterization of NPM1-mutated-specific T cells, stimulated for 3 hours with the 18 NPM1-mutated peptide mixture, was carried out on 33 (16 PB and 17 BM) samples obtained from 18 patients (Table 1 ) with the cytokine secretion assay (CSA Detection Kit, Miltenyi Biotec, Italy) including the following cytokines: IFNγ, interleukin-2 (IL2) and tumor necrosis factor-α (TNFα), as previously described [13 (link), 14 (link)]. The memory phenotype of the cytokine-producing cells was assessed after sample counterstaining with mouse anti-human monoclonal antibody conjugates, as detailed in Supplementary methods. In addition, the cytotoxic phenotype of either CD8+ or CD4+ IFNγ-secreting T cells was assessed by using a monoclonal antibody against the degranulation marker CD107a. Results were expressed as CD8+ or CD4+ T cell percentages [13 (link), 14 (link)].
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