Whole blood samples were collected in EDTA tubes by peripheral venepuncture and then immediately delivered to the laboratory at ambient temperature. Preanalytical samples were stored at 4°C until processing began within 4 h. Whole blood was stained with DuraClone Immuno-monitoring panels (Duraclone IM phenotyping BASIC Tube, B53309; Duraclone IM T cell Subsets Tube, B53328; Duraclone IM TCRs Tube, B53340; Duraclone IM Treg Tube, B53346; Duraclone IM B cells Tube, B53318; Duraclone IM Dendritic Cell Tube, B53351; all from Beckman Coulter, Krefeld, Germany) according to the manufacturer’s recommendations or antibodies listed in Table 1 . Data were recorded with a Navios™ cytometer running Cytometry List Mode Data Acquisition and Analysis Software for Navios™ cytometer, version 1.3 from Beckman Coulter. Blinded analyses were performed using Kaluza version 1.3 by an experienced operator and verified by a second experienced operator.
Duraclone im phenotyping basic tube
Manufactured by Beckman Coulter
Sourced in Germany
The DuraClone IM Phenotyping BASIC tube is a laboratory equipment product designed for immunophenotyping applications. It provides a standardized and reliable method for the identification and enumeration of specific cell populations in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Navios cytometer, by Beckman Coulter (2 mentions)
Duraclone im t cell subsets tube, by Beckman Coulter (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Versalyse reagent, by Beckman Coulter (1 mentions)
Cell strainer, by BD (1 mentions)
Gallios instrument, by Beckman Coulter (1 mentions)
Cd16 pb, by Beckman Coulter (1 mentions)
Cytometry list mode data acquisition and analysis software version 1, by Beckman Coulter (1 mentions)
Cd45ra fitc, by Beckman Coulter (1 mentions)
Kaluza software version 2, by Beckman Coulter (1 mentions)
Cd45 kro, by Beckman Coulter (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Cyclosporin, by Merck Group (1 mentions)
6 protocols using duraclone im phenotyping basic tube
1
Comprehensive Immune Cell Profiling from Whole Blood
2
Mouse Splenic Lymphocyte Phenotyping
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Mouse splenic tissue was dissected under deep isoflurane anesthesia and homogenized with 5 mL chilled PBS. The suspension was passed through a cell strainer (100 µm; Falcon). The filtered cell suspension was centrifuged, and the cell pellet was washed twice in PBS. The final pellet was resuspended in 100 µL PBS and added to a DuraClone IM Phenotyping BASIC tube (Beckman Coulter). Mouse splenic lymphocytes were treated with PECy5 anti‐human CD45 antibody (HI30; BioLegend), PECy7 anti‐human CD3 antibody (SK7; BioSource), and FITC anti‐human CD19 antibody (J3‐119; Beckman Coulter) for 20 min on ice followed by perm/wash buffer (BioSource) for permeabilization. Cells were stained with PE anti‐human STING antibody (T3‐680; BioSource). After staining in accordance with the manufacturer's instructions, the cells were exposed to VersaLyse reagent (Beckman Coulter) for 15 min for red blood cell hemolysis, before being fixed with 4% paraformaldehyde in PBS. Lymphocyte phenotyping was performed using flow cytometry with the Gallios instrument (Beckman Coulter).
3
Flow Cytometric Analysis of CSF Cells
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CSF cells were quantified by flow cytometry in a subset of patients (Table 2 ). Cells were pelleted by centrifugation (300× g), resuspended in 100 uL flow buffer (PBS containing 1% bovine serum albumin, 2 mM EDTA, and 2 mM sodium azide), applied to a DURAclone IM Phenotyping BASIC tube (Beckman Coulter, Brea, CA, USA), mixed, and incubated at 4 °C for 30 min. The cells were washed in flow buffer and fixed with 100 µL 2% paraformaldehyde. Data were acquired using the CytoFLEX (Beckman Coulter, Brea, CA, USA), and all cells in the sample were quantified.
4
PBMC Immunophenotyping by Flow Cytometry
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PBMC flow cytometry was performed on a majority subset of the cohort based on sample availability. PBMCs (200,000 cells) were stained with 1µL LIVE/DEAD™ fixable violet stain (Invitrogen/Thermo) in 1mL PBS and incubated for 30 minutes at 4°C in the dark. The cells were washed with 4mL PBS containing of 1% bovine serum albumin, 2mM EDTA, and 2mM sodium azide. Cells were decanted and resuspended in 100µL and added to a DURAclone IM Phenotyping BASIC tube (Beckman Coulter), mixed and incubated at 4°C for 30 minutes. The cells were washed and fixed with 100µL 2% paraformaldehyde. Data was acquired using the CytoFLEX (Beckman Coulter).
5
Detailed Blood Sample Preparation and Analysis by Flow Cytometry
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Detailed step-by-step protocols for preparation and analysis of blood samples by flow cytometry are available as Supplementary Methods
6
EBV Transformation Assay with STING Inhibitor
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B cells were isolated from peripheral blood or CBMCs in accordance with the manual, using the EasySep/RosetteSep Human B Cell Enrichment Cocktail (StemCell Technologies). To confirm cell isolation, cells stained in a DuraClone IM Phenotyping BASIC tube (Beckman Coulter) were analyzed by FACS analysis (Beckman Coulter; Gallios). PBMCs and B cells were seeded into a 96‐well plate and infected with a 10‐fold serial dilution of virus fluid obtained from AGS EBV cells. PBMCs were cultured in the presence of cyclosporin (0.5 µg/mL; Sigma‐Aldrich). Medium exchange was performed every 4 d with medium containing a STING inhibitor (C‐176; 0.5 µM). At 3 wk after infection, the number of wells in which transformed cells were present was counted, and the 50% transforming dose (TD50) was calculated. Fluorescence images were obtained by fluorescence microscopy (Axio Observer 7; Zeiss).
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