Countbright beads

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

CountBright beads are fluorescently-labeled microparticles designed for use in flow cytometry applications. They provide a standardized method for quantifying the number of target analytes in a sample.

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Lsrfortessa, by BD (7 mentions) Golgiplug, by BD (6 mentions) Cytoflex, by Beckman Coulter (5 mentions) Lsrfortessa flow cytometer, by BD (4 mentions) 7 aad, by BD (4 mentions) Ionomycin, by Merck Group (4 mentions) Lsr 2, by BD (4 mentions) Facscanto 2, by BD (4 mentions) Cytoflex lx, by Beckman Coulter (3 mentions) Foxp3 fixation permeabilization buffer kit, by Thermo Fisher Scientific (3 mentions) Live dead viability dye, by Thermo Fisher Scientific (3 mentions) Lsr 2 flow cytometer, by BD (3 mentions) Cd20 e450 2h7, by Thermo Fisher Scientific (3 mentions) Facscanto 2 flow cytometer, by BD (3 mentions) Lsrii flow cytometer, by BD (3 mentions) Dnase 1, by Worthington (3 mentions) Ls magnetic column, by Miltenyi Biotec (3 mentions) Cytofix cytoperm kit, by BD (3 mentions) Golgistop, by BD (3 mentions) Anti cd8α clone 53 6, by BD (2 mentions)

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120 protocols using countbright beads

Quantifying Neuronal Nuclei in V1 Cortex

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Nuclei in samples from the flattened (right) V1 and processed using the flow fractionator were stained with DAPI and an antibody to NeuN and quantified by a flow cytometer (Collins et al., 2010 (link); Young et al., 2012 (link)). The flow fractionator also requires the addition of a known number of Countbright Beads (Invitrogen) (Young et al., 2012 (link)) (Figure 1). Specifically, free-floating, DAPI-stained or DAPI+/NeuN+ double-stained nuclei from the main suspension samples were counted in tandem with a fixed volume of fluorescent Countbright Beads (Invitrogen Inc.) using a Becton Dickson 5-laser LSRII flow cytometer (for details, see Collins et al., 2010 (link); Young et al., 2012 (link)) (Figure 1). All samples were vigorously mixed before counts were determined, and all counts were made in duplicate to assess variability. The flow cytometry technician was blind to the sample attributes prepared by DJM and to the manual count data. All flow cytometry experiments were conducted in the Vanderbilt University Medical Center Flow Cytometry Shared Resource. Each of the 61 samples from right V1 was processed in duplicate using the flow fractionator method.

Miller D.J., Balaram P., Young N.A, & Kaas J.H. (2014). Three counting methods agree on cell and neuron number in chimpanzee primary visual cortex. Frontiers in Neuroanatomy, 8, 36.

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Flow Cytometric Quantification of FluoSpheres

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FluoSpheres sampled on THOR or Coriolis were quantified on a flow cytometer using CountBright Absolute Counting Beads (Invitrogen). Briefly, a defined number of CountBright beads were added to 1 mL of sample, samples were acquired on a FACSCalibur (BD Biosciences) and analyzed on FlowJo (Tree Star). Sampling on THOR did not affect the integrity of Fluospheres, which were clearly detectable and distinguishable from counting beads (Data not shown). The concentration of FluoSpheres in the sample was calculated with CountBright beads according to the instructions of the manufacturer (Invitrogen).

Rufino de Sousa N., Sandström N., Shen L., Håkansson K., Vezozzo R., Udekwu K.I., Croda J, & Rothfuchs A.G. (2020). A fieldable electrostatic air sampler enabling tuberculosis detection in bioaerosols. Tuberculosis (Edinburgh, Scotland), 120, 101896.

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Multiparametric Flow Cytometry Analysis of T Cell Subsets

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The following fluorochrome-conjugated antibodies against surface and intracellular antigens were used at 0.25-1×10^–3 mg/ml: anti-Thy-1.1 (OX-7), anti-CD45.1 (A20), anti-KLRG1 (2F1), anti-CD25 (PC61), anti-CD62L (MEL-14), anti-IFN-γ (XMG1.2), anti-TNF (MP6-XT22), anti-CD44 (IM7), and anti-CD8α clone 53-6.7 (BD Biosciences). Cells were incubated with specific antibodies for 30 min on ice in the presence of 2.4G2 mAb to block FcγR binding. All samples were acquired with a FACS Canto II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (TreeStar). To determine cytokine expression, cellular suspensions containing T cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A (Leukocyte activation cocktail with Golgiplug; BD Biosciences) for 4 h. After stimulation, cells were stained an amine-reactive exclusion-based viability dye (Invitrogen) and antibodies against cell-surface antigens, fixed and permeabilized and intracellularly stained with specific anti-cytokine antibodies. Intranuclear staining for cytokines was carried out using the Foxp3 staining kit (eBioscience). Countbright beads (Invitrogen) were added for the flow cytometric quantification of absolute cell number.

Roychoudhuri R., Clever D., Li P., Wakabayashi Y., Quinn K.M., Klebanoff C.A., Ji Y., Sukumar M., Eil R.L., Yu Z., Spolski R., Palmer D.C., Pan J.H., Patel S.J., Macallan D.C., Fabozzi G., Shih H.Y., Kanno Y., Muto A., Zhu J., Gattinoni L., O’Shea J.J., Okkenhaug K., Igarashi K., Leonard W.J, & Restifo N.P. (2016). BACH2 regulates CD8+ T cell differentiation by controlling access of AP-1 factors to enhancers. Nature immunology, 17(7), 851-860.

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Quantifying Human Leukemia Cells in Mice

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Peripheral blood was obtained at the time of first AraC dose (day 0) and at the time of dissection (day 8) two days after the last dose of AraC to determine the fraction of human blasts using flow cytometry. NSG mice were humanely killed in accordance with European ethic protocols. Bone marrow (mixed from tibias and femurs) and spleen were dissected in a sterile environment and flushed in Hanks balanced salt solution with 1% FBS. MNCs from peripheral blood, bone marrow and spleen were labeled with FITC-conjugated anti-hCD3, PE-conjugated anti-hCD33, PerCP-Cy5.5-conjugated anti-mCD45.1, APC-conjugated anti-hCD45 and PeCy7-conjugated anti-hCD44 (all antibodies from Becton Dickinson, BD, except FITC-conjugated anti-hCD3 from Ozyme Biolegend) to determine the fraction of human blasts (hCD45⁺mCD45.1⁻hCD33⁺hCD44⁺ cells) using flow cytometry. Analyses were performed on a Life Science Research II (LSR II) flow cytometer with DIVA software (BD) or Cytoflex flow cytometer with CytoExpert software (Beckman Coulter). The number of AML cells/ul peripheral blood and number of AML cells in total cell tumor burden (in bone marrow and spleen) were determined by using CountBright beads (Invitrogen) using described protocol.

Farge T., Saland E., de Toni F., Aroua N., Hosseini M., Perry R., Bosc C., Sugita M., Stuani L., Fraisse M., Scotland S., Larrue C., Boutzen H., Féliu V., Nicolau-Travers M.L., Cassant-Sourdy S., Broin N., David M., Serhan N., Sarry A., Tavitian S., Kaoma T., Vallar L., Iacovoni J., Linares L.K., Montersino C., Castellano R., Griessinger E., Collette Y., Duchamp O., Barreira Y., Hirsch P., Palama T., Gales L., Delhommeau F., Garmy-Susini B., Portais J.C., Vergez F., Selak M.A., Danet-Desnoyers G., Carroll M., Récher C, & Sarry J.E. (2017). Chemotherapy Resistant Human Acute Myeloid Leukemia Cells are Not Enriched for Leukemic Stem Cells but Require Oxidative Metabolism. Cancer discovery, 7(7), 716-735.

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Murine Flow Cytometry Analysis

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For flow cytometry analysis, blood was obtained by cardiac puncture and collected in an EDTA-coated tube. Mice were subsequently perfused through the left ventricle with 10 mL cold PBS. Femur and spleen were collected. The infarcted heart tissue was collected and weighed. In Apoe^−/− mice, the aorta, from the aortic root to the iliac bifurcation, was also collected. Absolute cell counting was performed by adding CountBright beads (Invitrogen) to each sample and calculating the ratio of bead events to cell events. Data were acquired with an LSRFortessa flow cytometer (BD Biosciences) and were analyzed with the use of FlowJo v10.7.1.

Maier A., Toner Y.C., Munitz J., Sullivan N.A., Sakurai K., Meerwaldt A.E., Brechbühl E.E., Prévot G., van Elsas Y., Maas R.J., Ranzenigo A., Soultanidis G., Rashidian M., Pérez-Medina C., Heo G.S., Gropler R.J., Liu Y., Reiner T., Nahrendorf M., Swirski F.K., Strijkers G.J., Teunissen A.J., Calcagno C., Fayad Z.A., Mulder W.J, & van Leent M.M. (2023). Multiparametric Immunoimaging Maps Inflammatory Signatures in Murine Myocardial Infarction Models. JACC: Basic to Translational Science, 8(7), 801-816.

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Multicolor Flow Cytometry Analysis of Circulating B Cells

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Cells were analysed using seven‐colour direct immunofluorescence staining on a CytoFLEX LX Flow Cytometer (Beckman Coulter). Abs used were CD20‐AF700 (clone 2H7), CD27‐PE (M‐T271), CD38 PE‐Cy7 (HB7), CD24‐FITC (ML5) and IgD‐V500 (IA6‐2), all from BD Biosciences with CD138‐VioBlue (44F9) (Miltenyi). In a second panel, CD20, CD27, CD38, CD138 and IgG‐V500 (G18‐145); IgM‐APC (G20‐127) (both BD Biosciences); and IgA‐FITC (IS11‐8E10) (Miltenyi) were also tested on circulating B cells (day 0 only). Cell populations were gated on FSC and SSC plot to remove small debris events (including apoptotic bodies), while a total event gate recorded viable and dead cells (gated using the 7AAD dye 50 µg/ml [BD Biosciences]). Absolute cell counts were calculated using CountBright beads (25,000 beads/panel; Invitrogen). Analysis was performed with CytExpert software, version 2.3 (Beckman Coulter).
A classic gating strategy for positive/negative live cell events was used (see profile in Figure S1) to evaluate changes in marker positivity at days 0, 6 and 13 and reported as % of total live cells. A gating strategy to define circulating naïve/memory from B‐reg and EPB, and expression of surface Ig is described in Figure S2.

Xie X., Shrimpton J., Doody G.M., Conaghan P.G, & Ponchel F. (2021). B‐cell capacity for differentiation changes with age. Aging Cell, 20(4), e13341.

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Myeloid Cell Enumeration from Plasma

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To assess the number of myeloid cells from plasma incubation experiments, cells were stained with the following panel: CD3-APC (clone HIT3a), CD19-APC (clone HIB19), CD56-APC (clone 5.1H11), CD14-FITC (clone M5E2), CD15-AF700 (clone HI98), CD11b-PE-Cy7 (clone ICRF44), CD34-BV650 (clone 561), and CD38-PE/Cy5 (clone HIT2) (BioLegend). After staining, cells were resuspended in FACS buffer with 2% CountBright beads (Invitrogen) to allow determination of absolute counts during analysis. Flow cytometry data were acquired on a Cytoflex LX (Beckman Coulter) and analyzed using FlowJo v10.1.

Reyes M., Filbin M.R., Bhattacharyya R.P., Sonny A., Mehta A., Billman K., Kays K.R., Pinilla-Vera M., Benson M.E., Cosimi L.A., Hung D.T., Levy B.D., Villani A.C., Sade-Feldman M., Baron R.M., Goldberg M.B., Blainey P.C, & Hacohen N. (2021). Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro. Science Translational Medicine, 13(598), eabe9599.

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Comprehensive Cytometric Analysis of CAR T Cells

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All antibodies were titrated. CAR expression was measured with Alexa-Fluor-647–conjugated goat anti-mouse Fab (Jackson ImmunoResearch cat#115-607-003, RRID: AB_2338931). The following antibodies were used: CD19–PE (clone SJ25C1 BD Biosciences cat# 340364, RRID: 400018) or CD19–BUV395 (clone SJ25C1, BD Biosciences cat# 563551, RRID: AB_2738272), CD19–BV510 (clone SJ25C1, Biolegend cat#363020, RRID: AB_2564229), CD3–BUV737 (clone UCHT, BD Biosciences cat# 612750, RRID: RRID:AB_2870081), BUV-395CD4 (BD Biosciences cat# 563552, RRID:AB_2738273), and APC-cy7-CD8 (BD Biosciences cat# 557834, RRID:AB_396892). CountBright beads (Invitrogen cat# C36950) were used to determine the absolute number of cells according to the manufacturer’s protocol. 7-AAD or DAPI was used to exclude dead cells. Fc Receptor–Binding Inhibitor Antibody Human (eBioscience cat# 14-9161-73, RRID: AB_468582) was used to block Fc receptors. In vitro viability after RT was assessed using Zombie NIR (Biolegend cat# 423106) and Annexin V PerCP-Cy5.5 (Biolegend cat# 640936) according to the manufacturer’s protocols. Data were collected using BD LSR-II, BD LSR-Fortessa, and Cytek Northern Lights cytometers. Data were analyzed with FlowJo Software (Treestar/BD Biosciences, RRID: SCR_008520). Cell sorting was performed using a BD FACSAria cell sorter.

Kim A.B., Chou S.Y., Kang S., Kwon E., Inkman M., Szymanski J., Andruska N., Colgan C., Zhang J., Yang J.C., Singh N, & DeSelm C.J. (2023). Intrinsic tumor resistance to CAR T cells is a dynamic transcriptional state that is exploitable with low-dose radiation. Blood Advances, 7(18), 5396-5408.

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High-Throughput Flow Cytometry Analysis of PBMCs and BAL Samples

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Stained PBMCs or BAL-derived LMNCs were analyzed by the BD Fortessa SORP high-throughput system (HTS). Prior to the acquisition, CountBright Beads (Invitrogen) were added to each well and a fixed volume of the stained sample was acquired by HTS. For visualization of ex vivo stained total and CMV-specific CD8⁺ T cells (Fig. S1A), we used a compounded gating scheme previously described in (23 (link)) with a following sequential gate order: potential doublet exclusion based on a FSC-A and FSC-H plot, dead cell exclusion based on the live cell gate on FV-510 and FSC-A plot, inclusion of CD3⁺ cells based on a CD3⁺ gate with a CD14⁺CD16⁺CD19⁺ exclusion gate on a CD3-Alexa-700 and CD14CD16CD19-QDot605 plot, and a lymphocyte gate on an FSC and SSA log plot. CMV-Specific CD8⁺ T cells were gated further on MHC class I dextramer positive population on the MHC class I dextramer-PE and SSC-A plot (Supp. Fig. 1A). Data analysis and graphic representations were done with FlowJo v.10 (TreeStar, Ashland, OR).

Hoji A., Popescu I.D., Pipeling M.R., Shah P.D., Winters S.A, & McDyer J.F. (2019). Early KLRG1+ but not CD57+CD8+ T cells in Primary CMV Infection Predict Effector Function and Viral Control. Journal of immunology (Baltimore, Md. : 1950), 203(8), 2063-2075.

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Immune Cell Subsets Phenotyping by Flow Cytometry

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To minimize non-specific binding of antibodies to FcγR, cells were pre-incubated with anti-mouse CD16/32 (93; BioLegend) prior to performing surface staining. Immune cell subsets were defined based on following surface phenotypes: F4/80⁺CD11b⁺ CD11c⁻ (macrophages); CD335⁺ (NK cells); and CD11b⁺CD11c⁺B220⁻MHC-II⁺ (CD11b⁺ classical DC (cDC)). All cells were stained with 7-AAD (BioLegend) to exclude dead cells. CountBright beads (Invitrogen) were used to calculate absolute cell numbers by flow cytometry. Sample collection was performed on a BD Fortessa X-20 cytometer and data analyzed using FlowJo V.10.1r7 (Treestar, Ashland, OR, USA).

Jo S., Fotovati A., Duque-Afonso J., Cleary M.L., van den Elzen P., Seif A.E, & Reid G.S. (2020). Differential Depletion of Bone Marrow Resident B-ALL after Systemic Administration of Endosomal TLR Agonists. Cancers, 12(1), 169.

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