For DCs flow cytometry, PBMCs were centrifuged, pelleted and washed with Phosphate-buffered saline (PBS) and stained for 35 min at room temperature with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), BV421 CD86, BV650 CD11c, BV711 HLA-DR, BV786 CCR7 (CD197), FITC Lin-2 (CD3, CD14, CD19, CD20, CD56), BV605 CD16, PeCF594 PD-L1 (CD274), APC Integrin-β7 (BD Biosciences), PerCPCy5,5 CD4, APCCy7 CD1c, PeCy7 CD141 (BioLegend) and AF700 CD123 (R&D, San Diego, CA) antibodies. Then PBMCs were washed with Permeabilization Buffer 10X diluted 1:10 (eBioscience™), permeabilized by Fixation/Perm buffer (eBioscience™), and intracellularly stained with PE IDO (eBioscience, San Diego, CA, USA) antibody. DCs were gated based on Lin-2 HLA-DR expression. Each subset (mDCs and pDCS) was gated based on CD123 and CD11c expression. mDCs subsets were gated by using CD16, CD1c and CD141 staining, for gating strategy see Supplementary Fig. 1 . Flow cytometry analyses were performed on an LRS Fortessa flow cytometer using FACS Diva software (BD Biosciences). Data were analyzed using the FlowJo software (Treestar, Ashland, OR). At least 1 × 106 events were acquired per sample.
Pe ido
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PE IDO is a laboratory instrument designed for the measurement of indoleamine 2,3-dioxygenase (IDO) activity. IDO is an enzyme involved in the metabolism of the amino acid tryptophan. The PE IDO provides a quantitative analysis of IDO activity, which can be useful for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (1 mentions)
Lrsfortessa flow cytometer, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer 10x, by Thermo Fisher Scientific (1 mentions)
Fixation perm buffer, by Thermo Fisher Scientific (1 mentions)
Perm wash buffer, by BD (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
Ionomycin, by BioLegend (1 mentions)
Phorbol 12 myrstate 13 acetate pma, by BioLegend (1 mentions)
2 protocols using pe ido
1
Multiparametric Flow Cytometry for DC Subsets
2
Flow Cytometric Analysis of NK Cells
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All collected PF samples were centrifuged at 400 g at 4°C for 10 min, and then the supernatant was discarded. After incubation with allophycocyanin (APC)/cyanine 7(Cy7)-conjugated antihuman CD45, fluorescein isothiocyanate (FITC)-CD3, Brilliant Violet 421 (BV421)-CD56, APC-NKG2D, BV510-NKp46 and phycoerythrin-cyanine 7 (PE-Cy7)-NKp44, cells were fixed and permeabilized using Fixation/Permeabilization Solution and Perm/Wash Buffer (BD, San Diego, CA, USA) for further intracellular staining, including PE-IDO, BV510-IFN-γ, PE/Cy7-IL-10 and APC-IL-22 (all antibodies were purchased from eBioscience). Staining was performed at room temperature for 30 min. Finally, these cells were washed with PBS and analyzed by FCM (Becton Dickinson). NK cells cultured with ESCs for 5 days were collected and analyzed for the expression of IDO by FCM according to the above protocol. For intracellular cytokine analysis, brefeldin A (10 μg/mL, BioLegend), phorbol 12-myrstate 13-acetate (PMA) (50 ng/mL, BioLegend) and ionomycin (1 μg/mL, BioLegend) were added 4 h before flow cytometry.
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