Flow check

Manufactured by Beckman Coulter

Sourced in United States

The Flow-Check is a laboratory instrument designed to perform flow cytometry analysis. Its core function is to precisely measure and analyze the physical and chemical characteristics of particles or cells suspended in a fluid sample. The Flow-Check provides accurate data on parameters such as size, granularity, and fluorescence intensity of the analyzed samples.

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Flow-Check Fluorospheres (25 citations) Flow-Check Pro Fluorospheres (10 citations) Flow Check beads (5 citations) Flow Check Pro Beads (3 citations) Flow‐Check Pro (2 citations)

5 protocols using flow check

Basophil and Neutrophil Analysis by Flow Cytometry

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The flow cytometer was controlled daily using Flow‐Check (Beckman Coulter), and the stability of the compensations was examined daily using Flow‐Set (Beckman Coulter).
The absolute number of basophils and neutrophils was analysed. One hundred μl of whole blood was treated with the ImmunoPrep Reagent System (Beckman Coulter, USA) according to the manufacturer's instructions. Thereafter, the pretreated whole blood was mixed with 100 μl Flow‐Count beads (Beckman Coulter, USA), and the samples were analysed using flow cytometry, and the total number of leucocytes was counted.
Further, basophils were identified as CD203c+, CD193+ (Figure 1A,B) and neutrophils as CD15+, CD16+ (Figure 1C,D). Mean fluorescence intensity (MFI) was used to evaluate activation of basophils and neutrophils by expression of CD62L, CD11b and CD49d. In addition, basophil activation was also measured as MFI for CD203c and per cent positive CD63 basophils (%CD63+). A cut‐off for a negative test was set to a baseline CD63 expression of approximately 2·5%.

Kalm F., Mansouri L., Russom A., Lundahl J, & Nopp A. (2020). Adhesion molecule cross‐linking and cytokine exposure modulate IgE‐ and non‐IgE‐dependent basophil activation. Immunology, 162(1), 92-104.

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Platelet Activation Assay Protocol

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Vacutainer Citrate 4.5 mL tubes, Cellfix and antibodies PAC1-FITC, MsIgM-FITC and CD63-V450 BD were from BD Biosciences (Franklin Lakes, NJ, USA). Flow-Check, Flow-Set Fluorospheres and antibodies CD62P-PE, CD42b-APC and MsIgG-PE were from Beckman Coulter (Brea, CA, USA). Microtiter plates were from Bio-RAD (cat number CON960; CA, USA). Bovine serum albumin (BSA) was from Sigma-Aldrich (St Louis, MO, USA). ADP was from Sigma-Aldrich (St Louis, MO, USA), CRP-XL was purchased from Prof. Farndale (University of Cambridge, UK) and TRAP-6 was from Bachem (Bubendorf, Switzerland). Collagen and Epinephrine were from Chrono-Log Corp. (Haverton, PA, USA) and Ristocetin was from (American Biochemical & Pharmaceutical Corporation, Middlesex, U.K.). HEPES-Bovine serum albumin (BSA) buffer (20 mM Hepes, 137 mM NaCl, 2.7 mM KCL, 1 mM MgCl and 1% BSA).

Navred K., Martin M., Ekdahl L., Zetterberg E., Andersson N.G., Strandberg K, & Norstrom E. (2019). A simplified flow cytometric method for detection of inherited platelet disorders—A comparison to the gold standard light transmission aggregometry. PLoS ONE, 14(1), e0211130.

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Instrument Maintenance and Quality Control

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The instrument was maintained using daily cleaning procedures recommended by the manufacturer throughout the study period. The quality control used the Flow-Check and Flow-Set Pro Fluorospheres (Beckman Coulter) and the protocols used were set-up by the manufacturer's technical support engineers.

Pieper I.L., Radley G., Christen A., Ali S., Bodger O, & Thornton C.A. (2018). Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro. Artificial organs, 42(6).

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Immunophenotyping Bacteria using Flow Cytometry

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Samples were suspended in sterile saline solution with 5 % albumin to prevent non-specific antibody binding, then stained with (i) anti-human IgA or IgG labelled with FITC (Invitrogen catalog # A18782 and A18806); and (ii) the DNA-binding fluorophor SYTO62 (Invitrogen catalog # S11344) according to the manufacturer instructions. Anti-mouse IgA or IgG labeled with FITC (Invitrogen catalog # M31101 and A24525) were used for isotype controls.
Cell sorting was performed with the MoFloTM XDP flow cytometer (Beckman Coulter Inc.) using Argon 488 nm (blue) laser (200 mW power) and the 635 nm (red) diode laser (25 mW power) as light sources. The lasers were aligned using Flow-CheckTM (10 μm) and Flow-SetTM (3 μm) fluorospheres (Beckman Coulter, Inc.). Emission filters were 520/30 for FITC and 680/30 for SYTO62 respectively. Proper fluorescent labeling was assessed by fluorescence and confocal microscopy (Additional file 1: Figure S1). Cells were separated according to their fluorescence in both the FITC and SYTO62 channels (Ig-coated bacteria) or the SYTO62 channel only (non-coated bacteria).

Simón-Soro Á., D’Auria G., Collado M.C., Džunková M., Culshaw S, & Mira A. (2015). Revealing microbial recognition by specific antibodies. BMC Microbiology, 15, 132.

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PIMA Analyzer Quality Control and Maintenance

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Quality control and routine PIMA™ analyser maintenance were performed daily as per manufacturer’s guidelines: one control has low CD4+ T-cell counts (115 cells/mm³ – 235 cells/mm³) and the other has normal CD4+ T-cell counts (719 cells/mm³ – 1355 cells/mm³). Daily quality control was conducted on all 3 analysers for the first 10 measurements when a new cartridge was used and over a period of 165 days (23 January – 25 March 2014). Accuracy and precision of the NHLS PLG testing was established in the NHLS laboratories by daily monitoring of instrument stability (Flow check TM, Beckman Coulter Miami, FL) and system performance verification using normal (394 cells/mm³ – 754 cells/mm³) and low (62 cells/mm³ – 206 cells/mm³) Immunotrol™ controls (Beckman Coulter, Miami, FL). The Addington NHLS laboratory participates in the NHLS proficiency testing panels and is accredited by the South African National Accreditation System.²⁷

Skhosana M., Reddy S., Reddy T., Ntoyanto S., Spooner E., Ramjee G., Ngomane N., Coutsoudis A, & Kiepiela P. (2016). PIMA™ point-of-care testing for CD4 counts in predicting antiretroviral initiation in HIV-infected individuals in KwaZulu-Natal, Durban, South Africa. Southern African Journal of HIV Medicine, 17(1), 444.

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