Flow set pro beads

Manufactured by Beckman Coulter

Sourced in United States

Flow-Set Pro Beads are a certified reference material used for the calibration and quality control of flow cytometry instruments. They provide a reliable and consistent way to verify the performance and sensitivity of flow cytometers.

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Flow set beads (2 citations) Flow‐Set (2 citations)

5 protocols using flow set pro beads

Comprehensive Flow Cytometry Protocol

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Blood samples were stained within 4 h and analyzed by flow cytometry according to the protocol of the ONE-Study Consortium (27 (link), 28 (link)). In addition, we included a chemokine receptor panel for categorization of T helper and Treg cell subsets (panel 6, see Supplementary Figure 1 for gating strategy). All fluorochrome-conjugated antibodies used are listed within Supplementary Table 1. In general, 100 μl EDTA blood were directly stained with prepared panel antibody mixes and incubated before lysing erythrocytes with lyse-fix solution composed of Versa Lyse™ and IOTest® Fixative Solution (Beckman Coulter GmbH). For the Treg panel (panel 7) 50 μl EDTA blood were used and additionally stained for intracellular expression of Foxp3 using the PerFix-nc Kit (Beckman Coulter), whereas for the B cell panel (panel 4) 300 μl EDTA blood was first lysed with Red Blood Cell Lysis Solution (Miltenyi Biotec GmbH) prior to antibody staining. The dendritic cell panel 5 was prepared twice and combined after staining. Samples were measured on a 10 color Navios flow cytometer (Beckman Coulter). Calibration with “Flow-Set Pro Beads” and “Flow Check Pro Beads” (both Beckman Coulter) was performed daily.

Stobutzki N., Schlickeiser S., Streitz M., Stanko K., Truong K.L., Akyuez L., Vogt K., Appelt C., Pascher A., Blau O., Gerlach U.A, & Sawitzki B. (2019). Long-Term Signs of T Cell and Myeloid Cell Activation After Intestinal Transplantation With Cellular Rejections Contributing to Further Increase of CD16+ Cell Subsets. Frontiers in Immunology, 10, 866.

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Multicolor Flow Cytometry Acquisition

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Sample acquisition was performed on 10-color, 3-laser NAVIOS flow cytometers (Beckman Coulter) using predefined settings. Debris was excluded by appropriate adjustment of the FSC recording trigger. Each sample was run twice in order to enhance the number of recorded events. Those duplicate readings were merged using the Kaluza analysis software prior to the final analysis.
Acquisition settings were defined according to the manufacturer’s instructions using the eight DuraClone RE PC compensation tubes as well as single CD117 or CD3 staining. Obtained photomultiplier tube (PMT) voltages were used to define target channels for all scatter and fluorescence detectors using calibration bead particles (Flow-Set Pro beads, Beckman Coulter). Matching of target channels was verified daily with a new calibration run to prevent target mismatch. Furthermore, all instruments underwent daily verification of optical alignment and fluidics using other calibration bead particles (Flow Check beads, Beckman Coulter).

Blum A., Haussmann K., Streitz M., Schlickeiser S., Tietze-Buerger C., Blau I.W, & Uharek L. (2019). Standardized assay for assessment of minimal residual disease in blood, bone marrow and apheresis from patients with plasma cell myeloma. Scientific Reports, 9, 2922.

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Multicolor Flow Cytometry Immune Profiling

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Conjugated monoclonal antibodies purchased from Beckman Coulter were used in the following combinations; CD57-fluorescein isothiocyanate (FITC)/CD279-phycoerythrin (PE)/CD45RA- energy-coupled dye (ECD)/CD8-allophycocyanine (APC)/CD4- APC/Alexa700 (APC700)/CD3-APC/Alexa750 (APC750), CD8-APC/CD4-APC700/CD3-APC750/CD38-PE/HLADR-PE-Cyanine7 (PC7), CD3-APC750/CD16-APC/HLADR-PC7/CD56- PE-Cyanine5.5 (PC5.5)/CD57-FITC, CD14-PE/CD16-APC.
Whole blood collected on EDTA was labeled and stored in the dark for 10 minutes at room temperature and fixed (IMMUNOPREP reagent system kit and TQ Prep automate, Beckman Coulter) according to the manufacturer’s recommendations. Samples were run on a Navios flow cytometer (Beckman Coulter) and results were analyzed using Kaluza software (Beckman Coulter). A minimum of 20,000 lymphocytes were analyzed per subpopulation. The flow cytometer’s inter-run stability was verified using the same batch of FlowSet Pro Beads (Beckman Coulter).

Cezar R., Kundura L., André S., Lozano C., Vincent T., Muller L., Lefrant J.Y., Roger C., Claret P.G., Duvnjak S., Loubet P., Sotto A., Tran T.A., Estaquier J, & Corbeau P. (2024). T4 apoptosis in the acute phase of SARS-CoV-2 infection predicts long COVID. Frontiers in Immunology, 14, 1335352.

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Multicolor Flow Cytometry Immunophenotyping

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Monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), energy-coupled dye (ECD), PE-Cyanine5.5 (PC5.5), PE-Cyanine7 (PC7), allophycocyanine (APC), APC/Alexa700, or APC/Alexa750 were purchased from Beckman Coulter. The antibodies were used in the following combinations; CD57-FITC/CD279-PE/CD45RA-ECD/C2-PC5.5/CD27-PC7/CD8-APC/CD4-APC700/CD3-APC750, CD8-APC/CD4-APC700/CD3-APC750/CD38-PE/HLADR-PC7/CD20-FITC, CD3-APC750/CD16 APC/HLADR-PC7/CD56-PC5.5/CD69-PE/CD57-FITC, CD4-FITC/CD45RA-ECD/CD25 PC7/FOXP3-APC/CD127-APC750. Whole blood collected into EDTA tubes was stained within 1 hour for 10 min at room temperature in the dark with cocktail of antibodies and fixed using IntraPrep Permeabilization Reagent Kit (Beckman Coulter) according to manufacturer's protocol. Cells were run on a Navios flow cytometer and results were analyzed by using Kaluza software (Beckman Coulter). A minimum 20,000 lymphocytes were gated to analyze the subpopulations. Representative gating strategies and images of flow plots are shown in Supplementary Fig. 1. We controlled the inter-run variability with the same batch of FlowSet Pro Beads (Beckman Coulter). The targets were defined on the samples settings (voltages). During the study, no voltage adjustment has been necessary to keep the beads into their respective defined targets.

Psomas C., Younas M., Reynes C., Cezar R., Portalès P., Tuaillon E., Guigues A., Merle C., Atoui N., Fernandez C., Le Moing V., Barbuat C., Marin G., Nagot N., Sotto A., Eliaou J.F., Sabatier R., Reynes J, & Corbeau P. (2016). One of the immune activation profiles observed in HIV-1-infected adults with suppressed viremia is linked to metabolic syndrome: The ACTIVIH study. EBioMedicine, 8, 265-276.

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Flow Cytometry Analysis of Cell Surface Markers

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Flow cytometric fluorescent anti-human monoclonal cell surface antibody (dry powder) tubes (DuraClone IM) were purchased from Beckman Coulter (Bangalore, India). The details of every fluorochrome-conjugated antibody, the schemes of every fluorochrome channel and the compensation controls (each of a single color) are presented in Table 2. Briefly, 100 μL of anticoagulant blood was stained with fluorescent antibodies for 15 minutes in the dark (room temperature). The erythrocytes were removed by adding two mL of lyse-fix solution consisting of Versa Lyse™ and IOTest^VR Fixative Solution (MBL Life Science, Nagoya, Japan) and incubated for 15 min in the dark (room temperature). Then, the cells were rinsed twice and resuspended in staining buffer (phosphate-buffered saline containing 2% fetal bovine serum) prior to acquisition. All samples were analyzed with a 13-Color CytoFlex Flow Cytometer (Beckman Coulter, Brea, CA, USA) after daily calibration with Flow-Set Pro Beads (Beckman Coulter, Brea, CA, USA).

Zhuang Q., Peng B., Wei W., Gong H., Yu M., Yang M., Liu L, & Ming Y. (2019). The detailed distribution of T cell subpopulations in immune-stable renal allograft recipients: a single center study. PeerJ, 7, e6417.

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