PMBCs were isolated from heparinized blood by the density gradient centrifugation method with Histopaque (Sigma, St Louis, MO, USA). 2 × 106 cells were cultured with stimulated NECM for 72 h, and then IL-5, IFN-γ, and tumor necrosis factor (TNF)-α production was measured with an ELISA kit (R&D system). To determine ILC2 expression with flowcytometry, cells were stained as described previously [26 (link)]. PBMCs were stained with a FITC CD2, CD3, CD14, CD16, CD19, CD56, CD235 lineage cocktail (eBioscience, San Diego, CA, USA) and FcεRI. Moreover, they were further stained with phycoerythrin (PE)-conjugated chemoattractant receptor–homologous molecule expressed on Th2 lymphocytes (CRTH2) and PE-Cy7 conjugated CD127 (BD Biosciences, Franklin Lakes, NJ, USA), and fixed with 4% paraformaldehyde. ILC2s were identified as Lin-CRTH2+CD127+ lymphocytes with a flowcytometer machine (Beckman Coulter, Hercules, CA, USA).
Pe cy7 conjugated cd127
Manufactured by BD
Sourced in United States
PE-Cy7 conjugated CD127 is a laboratory reagent used for flow cytometry analysis. It is a fluorescently labeled antibody that binds to the CD127 protein, also known as the Interleukin-7 receptor alpha chain. This reagent can be used to identify and quantify cells expressing CD127 in various biological samples.
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3 protocols using pe cy7 conjugated cd127
1
Isolation and Analysis of ILC2 from PBMCs
2
Identification of Human and Murine ILC2s
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Lineage negative cells were enriched from PBMCs using FITC lineage cocktail (eBioscience, San Diego, CA) and FceRI (eBioscience). Cells were further stained with PE-conjugated CRTH2 (BD Bioscience, NJ) and PE-Cy7 conjugated CD127 (BD Bioscience, NJ). Human and mice ILC2s were identified as Lin−CRTH2+CD127+ and Lin−ST2+CD45+CD90.2+CD25+Sca1+ lymphocytes, respectively. Flow cytometry was performed by the Beckman flow cytometer machine (Beckman Coulter, Hercules, CA, USA).
3
Identification and Quantification of ILC2s
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ILC2s were analyzed according to the method reported by Mjösberg et al.[16 (link)] Briefly, peripheral blood cells were stained simultaneously using an antihuman fluorescein isothiocyanate-conjugated lineage cocktail, phycoerythrin (PE)-conjugated CRTH2, and PE-CY7-conjugated CD127, or appropriate isotype controls (all from BD Pharmingen, San Diego, CA, USA). The cell lineage cocktail comprised antibodies to CD3, CD16, CD14, CD19, CD34, CD123, CD11c, T-cell receptor (TCR) αβ, and TCRγδ expressed on T-cells, B-cells, monocytes, macrophages, mast cells, dendritic cells, and hematopoietic progenitor cells. Lymphocytes lacking any of these lineage markers, as well as expressing CRTH2 and CD127, were considered to be the ILC2 population [Figure 1 ]. Cell counts were performed using the FACSAria II flow cytometry device (BD Biosciences, San Diego, CA, USA), and all data were analyzed using the FACSDiva software (BD Biosciences). The proportion of ILC2s was expressed as a percentage of total lymphocytes.
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