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Flow count beads

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Flow-Count beads are a type of microsphere used in flow cytometry applications. They are designed to provide a consistent and precise number of particles for quantitative analysis. The core function of Flow-Count beads is to enable accurate cell counting and enumeration in flow cytometry experiments.

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Lab products found in correlation

Facscalibur flow cytometer, by BD (3 mentions) Immunoprep reagent system, by Beckman Coulter (2 mentions) Celltracker orange, by Thermo Fisher Scientific (2 mentions) 70 μm nylon cell strainer, by BD (2 mentions) Medimachine, by BD (2 mentions) Cellvue claret, by Merck Group (2 mentions) Recombinant chemokines, by Thermo Fisher Scientific (2 mentions) Cytomics navios, by Beckman Coulter (2 mentions) Facscanto 2, by BD (2 mentions) Navios flow cytometer, by Beckman Coulter (2 mentions) Navios, by Beckman Coulter (2 mentions) Kaluza software, by Beckman Coulter (2 mentions) Uti lyse, by Beckman Coulter (2 mentions) Flow check, by Beckman Coulter (1 mentions) Flow set, by Beckman Coulter (1 mentions) Anti cd61 pe, by Thermo Fisher Scientific (1 mentions) Tumor dissociation kit, by Miltenyi Biotec (1 mentions) Zombie yellow fixable viability kit, by BioLegend (1 mentions) Anti mouse cd16 32 2.4g2, by BD (1 mentions) Propidium iodide, by Merck Group (1 mentions)

Spelling variants of this product

Flow-Count (21 citations) Flow count calibrator beads (8 citations) Flow-Count Fluorosphere Beads (5 citations) Flow counts beads (2 citations)

30 protocols using flow count beads

1

Antiproliferative Potency of Novel Compounds

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Example 11

TABLE 7
Antiproliferative activity in human MT-2 leukemic cells in vitro
CC50, 50% Cytotoxic
concentration μMMT2
Compoundcells T cell leukemia
Bn-ODE-PMEG0.036 ± 0.04
Bn-ODE-PMEG slow<0.01
Bn-ODE-PMEG fast<0.01
Bn-ODE-PMEA<0.010

Method of cytotoxicity determination. MT-2 cells were incubated with drug for 72 hrs and harvested. Flow count beads (Beckman Coulter, Miami, Fla.) were added to the cell suspension followed by propidium iodide staining and analysis using flow cytometer and the 50% cytotoxic concentration (CC50) was calculated from the cell counts and viability.

Results. Compounds disclosed herein are effective antiproliferative agents in human T cell leukemia (MT-2) cells (Table 7).

US10195222B2. Acyclic nucleoside phosphonate diesters (2019-02-05). The Regents of the University of California [US]. Inventors: Karl Y. Hostetler [US], James R. Beadle [US], Nadejda Valiaeva [US].
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2

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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3

Quantifying Viable Cell Density

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To remove IL-3 from the original culture medium, the cells were washed two times by PBS(-) and then seeded into 24-well plates at a density of 1 × 105 cells/ml in the RPMI medium supplemented with/without 50 nM AP20187 under 37℃ in a 5% CO2 incubator. Viable cell density was measured after 3 day-incubation in flow cytometry as follows. The cell suspension (100 µl) was transferred to 96-well plates, in which 1 μg/ml propidium iodide, 4 µl Flow-Count beads (Beckman Coulter, Brea, CA), and 46 µl PBS were added to each well in advance. The number of cells was measured using a FACSCalibur flow cytometer (Becton–Dickinson, Franklin Lakes, NJ). Viable cells were identified as propidium iodide-negative cells using FlowJo software (Becton–Dickinson).
Kongkrongtong T., Zhang R, & Kawahara M. (2021). Rational design of heterodimeric receptors capable of activating target signaling molecules. Scientific Reports, 11, 16809.
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4

Cytotoxicity Determination in T-cell Leukemia

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Example 24

Method of Cytotoxicity Determination.

MT-2 cells were incubated with drug for 72 hrs and harvested. Flow count beads (Beckman Coulter, Miami, Fla.) were added to the cell suspension followed by propidium iodide staining and analysis using flow cytometer and the 50% cytotoxic concentration (CC50) was calculated from the cell counts and viability.

Results.

Compounds disclosed herein are effective antiproliferative agents in human T cell leukemia (MT-2) cells (Table 12).

TABLE 12
Antiproliferative activity in human MT-2 leukemic cells in vitro
CC50, 50% Cytotoxic concentration
Compound #μMMT2 cells T cell leukemia
10.036 ± 0.04
1a<0.01
1b<0.01
2<0.010

US10076533B2. Acyclic nucleoside phosphonate diesters (2018-09-18). The Regents of the University of California [US]. Inventors: Karl Y. Hostetler [US], James R. Beadle [US], Nadejda Valiaeva [US].
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5

Quantifying Platelet-like Particle Production

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Fixed cells or PLP were analyzed by flow cytometry using anti-CD61-PE (Invitrogen, Carlsbad, CA) antibodies. The PLP production capacity was defined using flow count beads (Beckman Coulter, Brea, CA) as an internal control. In total, 5 × 104 flow count beads were added to each sample. The number of YFP+CD61+ events with forward/side scatter (FSC/SSC) properties as human peripheral blood platelets per 2,000 units of flow count were enumerated. The number of PLP per cell was calculated by the following equation: PLP density (event/μl) = the concentration of flow count x number of PLP/number of beads x the volume of flow count/the volume of PLP suspension. Absolute number of PLP per YFP- and CD61-double-positive cells = PLP density x total PLP volume / number of plated YFP-positive cells. PLP size was recorded as the GeoMean of FSC in FACS analysis.
Daimon E., Shibukawa Y., Thanasegaran S., Yamazaki N, & Okamoto N. (2021). Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells. Scientific Reports, 11, 17990.
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6

Basophil Enumeration in Whole Blood

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The absolute number of basophils was estimated in 100 μL of whole blood by using the ImmunoPrep reagent system (Beckman Coulter) according to manufacturer's instructions. Subsequently, 100 μL of pretreated whole blood was mixed with 100 μL flow-count beads (Beckman Coulter) before flow cytometric analysis.
Aljadi Z., Mansouri L., Nopp A., Paulsson J.M., Winqvist O., Russom A., Ståhl M., Hylander B., Jacobson S.H, & Lundahl J. (2014). Activation of Basophils Is a New and Sensitive Marker of Biocompatibility in Hemodialysis. Artificial Organs, 38(11), 945-953.
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7

Competitive Homing and Trafficking Assay for T Cells

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Competitive short-term (1 h) homing assays were performed by co-mixing (1:1 ratio) 2.5 × 107 murine CD8+ T cells labeled with 5 - (and 6) - carboxyfluorescein diacetate succinimidyl ester (CFSE) with equal numbers of murine CD8+ T cells labeled with 5 - (and 6) - (4 -chloromethyl) benzoyl) amino) tetramethylrhodamine (CellTracker Orange, Invitrogen Life Technologies) prior to i.v. adoptive transfer. In homing studies using ex vivo activated human T cells, ~1.5 × 107 of CFSE- or CTO-labeled cells were mixed at equivalent numbers prior to injection into tumor-bearing mice. Tumor and spleen were collected 1 h later and mechanically dissociated by Medimachine (BD Biosciences) or passed through a 70 μm nylon cell strainer (BD Biosciences). Ratios of fluorescent adoptively-transferred cells were determined by flow cytometry, and total cell numbers were assessed using Flow Count Beads (Beckman Coulter)14 (link). In selected experiments intratumoral infiltration by OT-I cells was monitored using tracking dyes (CellVue Claret, Sigma Aldrich) or by congenic mismatch (CD45.1+ OT-I→CD45.2+ host) up to 3 weeks after transfer.
Mikucki M., Fisher D., Matsuzaki J., Skitzki J., Gaulin N., Muhitch J., Ku A., Frelinger J., Odunsi K., Gajewski T., Luster A, & Evans S. (2015). Non-redundant Requirement for CXCR3 Signaling during Tumoricidal T Cell Trafficking across Tumor Vascular Checkpoints. Nature communications, 6, 7458.
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8

Effector T Cell Chemotaxis Assay

Cited 1 time
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Chemotaxis of murine and human effector T cells was assayed in 24 well plates (5 μM pore size transwell insert with polycarbonate membranes; Corning). Media alone (RPMI1640 + 1%BSA) or media containing recombinant chemokines (Peprotech) was placed at the bottom of triplicate wells. Murine T cell migration was assessed at 10 nM murine CXCL10, 1 nM murine CCL2, and 10 nM murine CCL5; human T cell chemotaxis was determined at 100 ng/mL recombinant human or murine chemokine20 (link) unless otherwise indicated. 5 × 105 cells were fluorescently labeled with CFSE or CTO, placed on the transwell insert, and incubated at 37°C for 3 h. Cells in the bottom chamber were enumerated by flow cytometry using Flow Count Beads (Beckman Coulter). Spontaneous migration was subtracted from all conditions, and data are reported either as absolute number of cells migrated or as migration relative to WT T cells.
Mikucki M., Fisher D., Matsuzaki J., Skitzki J., Gaulin N., Muhitch J., Ku A., Frelinger J., Odunsi K., Gajewski T., Luster A, & Evans S. (2015). Non-redundant Requirement for CXCR3 Signaling during Tumoricidal T Cell Trafficking across Tumor Vascular Checkpoints. Nature communications, 6, 7458.
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9

Tumor-Infiltrating Cell Characterization

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Tumor-infiltrating cells were prepared with a tumor dissociation kit (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. The total number of live cells analyzed was matched between the groups with Flow Count beads (Beckman Coulter, Fullerton, CA, USA). A Zombie Yellow fixable viability kit (BioLegend, San Diego, CA, USA) was used to stain dead cells. The cells were then pretreated with purified anti-mouse CD16/32 (2.4G2) (BD Biosciences, NJ, USA), stained with antibodies, and analyzed with a CytoFLEX (V5-B5-R3 configuration, Beckman Coulter, Fullerton, CA, USA). The following fluorescent-labeled antibodies were purchased from BioLegend (San Diego, CA, USA) and used for analysis: Brilliant Violet 785-conjugated anti-CD45 (30-F11), Brilliant Violet 605-conjugated anti CD11b (M1/70), allophycocyanin (APC)-conjugated anti-F4/80 (BM8), phycoerythrin (PE)-Cyanine7-conjugated anti-CD86 (GL-1), PE-conjugated anti-CD206 (MMR), and fluorescein isothiocyanate (FITC)-conjugated anti-CD49b (HMα2). For all channels, positive and negative cells were gated on the basis of fluorescence minus one controls, and CD86 and CD206 were gated with appropriate isotype controls (Figure S1). The data were analyzed with FlowJo software v10.4.
Sugawara K., Iwai M., Yajima S., Tanaka M., Yanagihara K., Seto Y, & Todo T. (2020). Efficacy of a Third-Generation Oncolytic Herpes Virus G47Δ in Advanced Stage Models of Human Gastric Cancer. Molecular Therapy Oncolytics, 17, 205-215.
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10

Cell Proliferation Assay with Apoptosis Modulators

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Subcultured cells were washed with PBS twice to remove IL-3 in the culture medium. Cells were seeded into 24-well plates at 1 × 105 cells/ml with or without serial concentrations of AP20187 or Nutlin-3 (FUJIFILM Wako Pure Chemical) for 72 h. Flow cytometry was employed for cell counting. Before measurement, the sample mixtures (100 µl of cell suspension mixed with 4 µl of Flow-Count beads (Beckman Coulter, Brea, CA) and 46 µl of PBS containing 1 µg/ml of propidium iodide (Sigma‐Aldrich) at a final concentration) were transferred to 96-well plates and the number of cells was counted with a FACSCalibur flow cytometer (Becton–Dickinson, Franklin Lakes, NJ) calibrated by the count of Flow-Count beads. The data was processed by the software FlowJo v7.6.5 (Becton–Dickinson). The EC50 values of the helper ligand was calculated based on the results of the proliferation assay by curve fitting to the logistic function (Rodbard) using the software ImageJ v1.53 (National Institutes of Health, Bethesda, MD). The EC50 values were listed with the previously reported in vitro affinity values (Supplementary Fig. S3a).
Kashima D, & Kawahara M. (2021). Evolution of KIPPIS as a versatile platform for evaluating intracellularly functional peptide aptamers. Scientific Reports, 11, 11758.
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