Immunophenotyping of bone marrow (BM) was performed using an FC500 or Facscalibur flow cytometer. FACS buffer was made with PBS+2mM EGTA+2% FBS. Primary AML and leukemic stem cell fractions were detected using the following antibodies (company; product #; clone): anti-CD34 PE (BD bio-sciences; 348057; 8G12), anti-CD34 FITC (BD Pharmingen; 555821; 581), anti-CD38 APC (ebiosciences; 17-0389-42; HIT2) and anti-CD123 PE (BD Pharmingen; 558714; 7G3), anti-CD45 APC (BD Pharmingen; 557513; TU116) and anti-class I HLA A, B, C (Biolegend; 311404; W6/32). NK-92 cells lines were assessed for CD16 expression using CD16 PE (Biolegend; 302008; 3G8). Leukemia cell lines were evaluated using anti-CD123 PErCy5.5 (BD Biosciences; 560904; 7G3). Cell sorting was performed using a FacsAria cell sorter as described in the Online Supplementary Methods.
Anti cd123 pe
Manufactured by BD
Anti-CD123-PE is a fluorochrome-conjugated monoclonal antibody that binds to the CD123 antigen. CD123 is a marker expressed on the surface of certain cell types, including hematopoietic progenitor cells and some leukemic cells. The PE (phycoerythrin) fluorochrome is used for detection and quantification purposes in flow cytometry and other analytical techniques.
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Lab products found in correlation
Anti cd34 pe, by BD (1 mentions)
Anti cd45 apc, by BD (1 mentions)
Cd16 pe, by BioLegend (1 mentions)
Anti cd38 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd34 fitc, by BD (1 mentions)
Anti cd45 af700, by BD (1 mentions)
Anti igd pe cy7, by BD (1 mentions)
Anti hla dr percp cy5, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Fc blocking reagent, by Miltenyi Biotec (1 mentions)
Anti hla dr percp, by BD (1 mentions)
Anti cd33 apc, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
4 protocols using anti cd123 pe
1
Immunophenotyping of Bone Marrow Cells
2
Isolation of Human Basophils by Flow Cytometry
3
Basophil FcεRI Expression in CSU
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Two independent researchers evaluated the levels of basophil FcεRI receptor and the PROs. FcεRI receptor expression on basophils was measured by flow cytometry from peripheral blood samples obtained from patients with CSU on day 0 and on weeks 4, 8, 20 and 44 of treatment, and from healthy controls (HCs). Flow cytometry analysis was performed following standard procedures. Briefly, 150 μl anticoagulated blood was incubated on the same day of collection during 20 min at 4ºC, with an excess of human immunoglobulins to block unspecific binding. Afterwards, blood was stained with either anti-CD123-PE (BD) or anti-CD193-APC (Miltenyi) to identify basophils and with anti-FcεR1a-FITC (clone CRA1, Ebiosciences) or an isotype control to establish the expression of FcεRI on the surface of blood basophils. After incubation, red blood cells were lysed with a hypotonic solution and samples were analysed by flow cytometry using the FACSDiva software. At least 2 × 10 5 events were acquired. Gating strategy is represented in Fig. 1. Levels of basophil FcεRI receptor are expressed as mean fluorescence intensity (MFI).
4
Quantifying Circulating Monocyte HLA-DR and DC Subsets
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Quantifi cation of HLA-DR on circulating monocytes was performed using a standardised fl ow cytometric assay (QuantiBRITE HLA-DR, BD Biosciences, Heidelberg, Germany), as described elsewhere [17] . Enumeration of DC subsets was performed as described elsewhere [16] . In short, 150 μ L of whole blood was stained with fl ourescein isothiocyanate-conjugated antibodies against lineage (lin1) markers (mixture of anti-CD3/CD14/CD16/CD19/CD20/CD56), anti-CD123-PE, anti-HLA-DR-PerCP, and anti-CD33-APC (BD Biosciences, Heidelberg, Germany). PDC were gated as lin1-CD123 + HLA-DR + events and MDC as lin1-CD33 + HLA-DR + . Aft er treatment with FACS Lysing Solution, at least 300 events per DC population were analyzed on a FACSCalibur using CellQuest Pro (BD Biosciences, Heidelberg, Germany) software. HLA-DR expression on DCs was measured as mean fl uorescence intensity. Absolute APC population frequencies were calculated as WBC counts multiplied by the ratio of the APC population over all leukocytes. The gating strategy is given ( Figure 1 ).
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