Mixed disaggregated pieces of tumor were enzymatically digested at 37°C in RPMI 1640 with collagenase IV (2 mg/mL, Sigma), DNase (0.1 mg/mL, Sigma), and hyaluronidase (0.1 mg/mL, Sigma). Cell suspensions were passed through 100-μm filters to remove aggregates. Cells were washed with staining buffer (#420201, Biolegend) and stained with conjugated antibodies. Live cells were identified using LIVE/DEAD staining (#L23105, ThermoFisher). Flow cytometry analyses were performed using BD LSRFortessa. Conjugated antibodies were purchased from Biolegend: CD45 (clone 2D1), CD19 (clone 4G7), CD56 (clone 5.1H11), CD11b (clone ICRF44), CD16 (clone 3G8), CD66b (clone G10F5), HLA-DR (clone L243), CD14 (clone 63D3), and CD15 (clone HI98). Five × 106 total events were collected from each tumor sample to analyze a sufficient number of intracellular lymphocytes. Flow data were analyzed by FlowJo V10. Gating strategies were determined by fluorescence minus one (FMO). Tumor-infiltrated dendritic cells (TIDC) were sorted and cultured in complete 1640 medium without further expansion.
Cd15 clone hi98
Manufactured by BioLegend
Sourced in United States
CD15 (clone HI98) is a monoclonal antibody that binds to the CD15 antigen, also known as Lewis X or stage-specific embryonic antigen 1 (SSEA-1). CD15 is a carbohydrate antigen expressed on the surface of various cell types, including granulocytes, monocytes, and some stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Staining buffer, by BioLegend (1 mentions)
Live dead staining, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Lysis buffer, by Qiagen (1 mentions)
Facs cantotm 2 flow cytometer, by BD (1 mentions)
Cd62l clone dreg 56, by BioLegend (1 mentions)
Foxp3 staining set, by Thermo Fisher Scientific (1 mentions)
Cd4 clone okt4, by BioLegend (1 mentions)
Cd33 clone wm53, by BioLegend (1 mentions)
Cd8 clone hit8a, by BioLegend (1 mentions)
Live dead fixable aqua dead cell kit, by Thermo Fisher Scientific (1 mentions)
Cd14 clone 61d3, by Thermo Fisher Scientific (1 mentions)
Hla dr clone ln3, by BioLegend (1 mentions)
Facsdiva, by BD (1 mentions)
3 protocols using cd15 clone hi98
1
Tumor-Infiltrated Dendritic Cell Isolation
2
Whole Blood Immune Cell Phenotyping
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Whole blood was stained with anti-human CD14 (clone 61D3, Invitrogen, USA), CD15 (clone HI98, BioLegend, USA), CD16 (clone CB16, Invitrogen), CD62L (clone DREG-56, BioLegend, USA), and CD64 (clone 10.6, BioLegend, USA). After 15 min incubation in the dark, erythrocytes were lysed with 10x diluted Lysis Buffer (QIAGEN, Netherlands), and samples were washed twice with PBS. Samples were measured on BD FACS CantoTM II Flow Cytometer (BD Biosciences, USA) equipped with the BD FACS Diva Software v6.1.2 and analyzed with FlowJo v10.10.0. (FlowJo LLC, USA) software. Gating strategy is shown in Supplementary Figures 1 2
3
Profiling Peripheral Blood Immune Cells
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Peripheral blood samples were collected before the first treatment cycle. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood on Ficoll-Hypaque gradients as previously described [23] (link). Fresh PBMCs from patients were stained with antibodies targeting CD4 (clone OKT4; Biolegend), CD8 (clone HIT8a; Biolegend), CD25 (clone BC96, Invitrogen), CD127 (clone ebioRDR5; Thermo Fisher), CD3 (clone UCHT1; Thermo Fisher), CD19 (clone HCD56; Biolegend) and CD56, CD33 (clone WM53, Biolegend), HLA-DR (clone LN3; Biolegend), CD14 (clone 61D3, Thermo Fisher), and CD15 (clone HI98; Biolegend). Intracellular staining for anti-human FoxP3 (clone PCH101; Thermo Fisher) was done after fixation and permeabilization with the FoxP3 staining set (Thermo Fisher).
Isotype controls were used as negative controls. Dead cells were eliminated using the Live/Dead Fixable Aqua Dead Cell Kit (Thermo Fisher). Stained cells were analyzed on a LSRII cytofluorometer using FACS Diva (Becton Dickinson) and Flow-Jo Software (TreeStar).
Isotype controls were used as negative controls. Dead cells were eliminated using the Live/Dead Fixable Aqua Dead Cell Kit (Thermo Fisher). Stained cells were analyzed on a LSRII cytofluorometer using FACS Diva (Becton Dickinson) and Flow-Jo Software (TreeStar).
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