Counting beads

Manufactured by BioLegend

Sourced in United States

Counting beads are small, uniform particles used for quantitative analysis in flow cytometry and other applications. They provide a known number of particles per volume, allowing for accurate cell counting and quantification.

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Lab products found in correlation

Facscanto 2, by BD (5 mentions) Cxcl5, by Thermo Fisher Scientific (2 mentions) Cxcl2, by Thermo Fisher Scientific (2 mentions) Anti mouse ly6g 1a8, by BioLegend (2 mentions) Ls columns of macs cell separation system, by Miltenyi Biotec (2 mentions) 5 μm pore transwells 96 well plate, by Corning (2 mentions) Sav pe cy7, by BioLegend (2 mentions) Cxcl1, by Thermo Fisher Scientific (2 mentions) Collagenase type 1, by Worthington (1 mentions) Lsrii cytometer, by BD (1 mentions) Kaluza software, by Beckman Coulter (1 mentions) Costar transwell, by Corning (1 mentions) Facsdiva software, by BD (1 mentions) 70 μm cell strainer, by BD (1 mentions) Percoll gradient, by GE Healthcare (1 mentions) Fc blocker, by BioLegend (1 mentions) 7 aminoactinomycin d 7 aad, by BD (1 mentions) Brefeldin a, by BioLegend (1 mentions) Fixation permeabilization kit, by BD (1 mentions) Fluorofix buffer, by BioLegend (1 mentions)

Spelling variants of this product

Precision Counting Beads (18 citations)

15 protocols using counting beads

Adipose tissue macrophage phenotyping

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SVF cells were isolated from eWAT following digestion with collagenase type I (Worthington Biochemical Corporation). Cells were stained with Fc receptor blockade and antibodies to immune cell markers CD45 (Thermo Fisher #17-0451-82; RRID:AB_469392), F4/80 (Thermo Fisher #45-4801-82, RRID:AB_914345), CD11c (Thermo Fisher #12-0114-82, RRID:AB_465552), and CD206 (Bio-Rad Laboratories #MCA2235F, RRID:AB_324594) or respective isotype controls. Data were collected with an LSRII cytometer (BD Biosciences) and analyzed using Kaluza software (Beckman Coulter, Indianapolis, IN). Gating was performed on viable CD45+ cells, from which total macrophages were identified as F4/80+ high. Macrophages were classified as CD11c⁺/CD206⁻ “M1-like” macrophages and CD206⁺/CD11c⁻ “M2-like” macrophages. Data were presented as cell numbers per gram adipose tissue calculated using counting beads (Biolegend #424902) or as percentages of total macrophages and SVF.

Cox A.R., Masschelin P.M., Saha P.K., Felix J.B., Sharp R., Lian Z., Xia Y., Chernis N., Bader D.A., Kim K.H., Li X., Yoshino J., Li X., Li G., Sun Z., Wu H., Coarfa C., Moore D.D., Klein S., Sun K, & Hartig S.M. (2022). The rheumatoid arthritis drug auranofin lowers leptin levels and exerts anti-diabetic effects in obese mice. Cell metabolism, 34(12), 1932-1946.e7.

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Assessing T Cell Migratory Potential

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Following transduction, migratory potential of T cells was analyzed in a chemotaxis assay using 24-well culture plates carrying polycarbonate membrane-coated transwell permeable inserts of 5 μm pore size (3421, Costar Transwell, Corning). 2x10⁵ TILs or control T cells were seeded in the upper wells, and lower wells contained Lan1, SKNDZ or IMR-32 overnight cultures of 0.5x10⁶ tumor cells or 600 μL of complete media negative control. Cell migration was measured after 4 h or 24 h at 37°C and 5% CO₂ using counting Beads (424902, BioLegend).

Ollé Hurtado M., Wolbert J., Fisher J., Flutter B., Stafford S., Barton J., Jain N., Barone G., Majani Y, & Anderson J. (2019). Tumor infiltrating lymphocytes expanded from pediatric neuroblastoma display heterogeneity of phenotype and function. PLoS ONE, 14(8), e0216373.

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Isolation and Analysis of Infiltrating T-cells in Murine Organs

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Whole body perfusion with phosphate-buffered saline (PBS) was performed on human STAT5B^N642H or wild-type mice, or transplant recipient mice, and organs were then collected and minced through a 70 μm cell strainer (BD Biosciences). For organ T-cell infiltration experiments, leukocytes were isolated using 40% and 78% Percoll gradients (GE Healthcare). Cells were then stained for FACS analysis using various antibodies purchased from eBioscience, Biolegend or BD Biosciences (see Supplementary Table 3 for antibody list). Counting beads (BioLegend) were added before acquisition to quantify absolute cell numbers. All analyses were performed on a FACSCanto II flow cytometer using FACSDiva software (BD Biosciences). Further analyses were performed using FlowJo software. The gating strategies employed are shown in Supplementary Figs. 9 and 10.

de Araujo E.D., Erdogan F., Neubauer H.A., Meneksedag-Erol D., Manaswiyoungkul P., Eram M.S., Seo H.S., Qadree A.K., Israelian J., Orlova A., Suske T., Pham H.T., Boersma A., Tangermann S., Kenner L., Rülicke T., Dong A., Ravichandran M., Brown P.J., Audette G.F., Rauscher S., Dhe-Paganon S., Moriggl R, & Gunning P.T. (2019). Structural and functional consequences of the STAT5BN642H driver mutation. Nature Communications, 10, 2517.

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Multicolor Flow Cytometry for Immune Cell Profiling

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Enumeration of peripheral blood T, B and NK cells was undertaken using multicolor flow cytometry^{32 (link)}. Thawed PBMCs were washed twice in RPMI and suspended in FACS buffer and fluorescent-conjugated antibodies were added for 30 min at 2–8 °C before two washes and collected on a FACSCanto II (BD Biosciences). Data were analyzed using Cytobank.
PBMCs were analyzed for B cells (anti-CD19-PE-Cy7 and anti-CD3-FITC), CD4⁺ T cells (anti-CD3-FITC and anti-CD4-BV510), CD8⁺ T cells (anti-CD3-FITC and anti-CD8-APC eF780) and NK cells (anti-CD3-FITC and anti-CD56-PE) and markers of activation (HLA-DR-PerCP-Cy5.5 and CD38 APC). Counting beads (BioLegend) were used as per manufacturer’s instructions for cell counting.

Lim S.H., Stuart B., Joseph-Pietras D., Johnson M., Campbell N., Kelly A., Jeffrey D., Turaj A.H., Rolfvondenbaumen K., Galloway C., Wynn T., Coleman A.R., Ward B., Long K., Coleman H., Mundy C., Bates A.T., Ayres D., Lown R., Falconer J., Brake O., Batchelor J., Willimott V., Bowzyk Al-Naeeb A., Robinson L., O’Callaghan A., Collins G.P., Menne T., Faust S.N., Fox C.P., Ahearne M., Johnson P.W., Davies A.J, & Goldblatt D. (2022). Immune responses against SARS-CoV-2 variants after two and three doses of vaccine in B-cell malignancies: UK PROSECO study. Nature Cancer, 3(5), 552-564.

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Flow Cytometry Immunophenotyping Protocol

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For flow cytometric analysis, cells were stained with e506 LIVE/DEAD fixable dye (eBioscience) or 7-aminoactinomycin D (7-AAD) (BD Pharmigen) for 10 min on ice. Pelleted cells were incubated with Fc blocker (BioLegend) or normal mouse immunoglobulin G (IgG) (Life Technologies) for 10 min on ice to block the binding to Fc receptors. Extracellular antigens were stained for 20 min with antibodies on ice in a staining buffer. Cells were fixed and permeabilized with BD Cytofix/Cytoperm (for cytokine analysis) or eBioscience Transcription Factor Fix/Perm (for Bat3 or FoxP3 analysis) per the manufacturers’ instructions. Intracellular antigens were stained for 45 min on ice in the appropriate 1× perm/wash buffer before acquisition on a BD LSR II/Fortessa/Symphony flow cytometer (Becton Dickinson). The following antibodies were used (BioLegend unless otherwise specified): Bat3 (EPR9223, Abcam), anti-BrdU (3D4), FoxP3 (FJK-16, eBioscience), Granzyme B (QA16A02), IFN-γ (XMG1.2), IL-17A (Tc11–538 18H10.1), IL-10 (JES5–16E3), IL-2 (JES6–5H4), and TNF-α (MP6-XT22). Negative staining was determined using the appropriate isotype control antibody or Fluorescence Minus One control. Counting beads (BioLegend) were added to quantify absolute cell numbers. Results were acquired with the Diva software and analyzed using FlowJo software (Treestar).

Tang R., Acharya N., Subramanian A., Purohit V., Tabaka M., Hou Y., He D., Dixon K.O., Lambden C., Xia J., Rozenblatt-Rosen O., Sobel R.A., Wang C., Regev A., Anderson A.C, & Kuchroo V.K. (2022). Tim-3 adapter protein Bat3 acts as an endogenous regulator of tolerogenic dendritic cell function. Science immunology, 7(69), eabm0631.

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Multiparametric Flow Cytometry Analysis

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Cells were incubated in CD16/32 (Biocell #BE0307) and stained with antibodies as documented in SI Appendix, Table S1. Samples were fixed using Fluorofix buffer (Biolegend #422101). Intracellular staining was performed using a BD Biosciences Fixation/Permeabilization kit (BD Biosciences #554714) after stimulating cells for 4 h with Brefeldin A (Biolegend #420601). Absolute cell counts were determined using counting beads (Biolegend #424902). Cells were acquired on an LSRII (BD Biosciences) or an Aurora (Cytek) cytometer and analyzed with FlowJo software.

Gao K.M., Motwani M., Tedder T., Marshak-Rothstein A, & Fitzgerald K.A. (2022). Radioresistant cells initiate lymphocyte-dependent lung inflammation and IFNγ-dependent mortality in STING gain-of-function mice. Proceedings of the National Academy of Sciences of the United States of America, 119(25), e2202327119.

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Isolation and Characterization of Murine Neutrophils

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For BM neutrophils isolation, BM cells were stained with anti-mouse Ly6G (1A8;Biolegend) followed by purification using antibiotin magnetic beads and LS columns of MACS cell separation system (both Miltenyi Biotec). For in vitro infection, 10⁵ neutrophils were seeded per well and infected with Mtb HN878 at MOI 2 or 10. After 3 h of infection, cells were recovered for RNA analysis or reactive oxygen species detection with DHE and DHR probes. Cells were incubated with DHE or DHR at 37 °C for 10 or 30 min, respectively. Samples were then acquired on a BD FACS Canto II to detect red-fluorescence (DHE) or green fluorescence (DHR) due to probes oxidization. For the chemotaxis assay, 10⁵ neutrophils were seeded on the upper chamber of the 5 μm-pore transwells (96 well plate;Corning) and allowed to migrate for 3 h towards the lower chamber containing media with CXCL1, CXCL2 and CXCL5 (all from Peprotech) at 500, 50, 5 and 0.5 ng/ml or without cytokines. As control for the amount of cells seeded, cells were also seeded in the lower chamber. Cells were then recovered and stained with SAV PE-Cy7 (Biolegend). To quantify the number of migrated cells, counting beads (Biolegend) were added to the cells prior to acquisition in a BD FACS Canto II. All cytometry data were analysed using FlowJo version 10.1.r7.

Fonseca K.L., Maceiras A.R., Matos R., Simoes-Costa L., Sousa J., Cá B., Barros L., Fernandes A.I., Mereiter S., Reis R., Gomes J., Tapia G., Rodríguez-Martínez P., Martín-Céspedes M., Vashakidze S., Gogishvili S., Nikolaishvili K., Appelberg R., Gärtner F., Rodrigues P.N., Vilaplana C., Reis C.A., Magalhães A, & Saraiva M. (2020). Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils. Mucosal Immunology, 13(5), 836-848.

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Naive CD4 T Cell Skewing Protocol

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Naive CD4 cells were isolated from mice splenocytes (StemCell). Cells were activated for 3 days using immobilized anti-CD3 (10 μg ml⁻¹) and soluble anti-CD28 (2 μg ml⁻¹). Skewing conditions were as follows: T_H1, 1 μg ml⁻¹ anti-IL4, 5 ng ml⁻¹ IL2, and 10 ng ml⁻¹ IL12; T_H2, 1 μg ml⁻¹ anti-IFN-γ, 5 ng m^l−1 IL2, and 10 ng ml⁻¹ IL4; T_reg, 1 μg ml⁻¹ anti-IFN-γ, and 1 μg m^l−1 anti-IL4, and 2 ng ml⁻¹ TGFβ1. Bead-based multianalyte flow immunoassays (BD Bioscience) were used to measure cytokine production in the supernatant per manufacturer’s instruction. Absolute cell numbers were quantified with flow cytometry using counting beads (Biolegend).

Ho W.S., Wang H., Maggio D., Kovach J.S., Zhang Q., Song Q., Marincola F.M., Heiss J.D., Gilbert M.R., Lu R, & Zhuang Z. (2018). Pharmacologic inhibition of protein phosphatase-2A achieves durable immune-mediated antitumor activity when combined with PD-1 blockade. Nature Communications, 9, 2126.

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Peritoneal Immune Cell Characterization

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Peritoneal lavage exudate was collected by injecting 5 ml of 2 mM of EDTA in PBS into the peritoneal cavity. After gentle massaging, ~4 ml of exudate was removed with an 18G needle. Cells were washed in FACS buffer (0.05 % BSA, 2 mM EDTA in PBS pH 7.4) and then blocked using anti-CD16/32 (Biolegend) for 10 min at 4°C. Peritoneal cells were analyzed using anti-CD45 (clone 30-F11; BioLegend), anti-CD11b (clone M1/70; BioLegend), anti-F4/80 (clone BM8; BioLegend), anti-Ly6G (clone 1A8; BioLegend), anti-CD206 (clone C068C2; BioLegend), and anti-MHCII (clone. M5/114.15.2; BioLegend) antibodies. Absolute cell count was calculated by the addition of counting beads (BioLegend). Data were acquired using BD LSR II Fortessa (Becton Dickinson) and analyzed using FlowJo analysis software (version 10.6, Treestar Inc.). The gating strategy is depicted in Figure S1.

O'Riordan C.E., Purvis G.S., Collotta D., Krieg N., Wissuwa B., Sheikh M.H., Ferreira Alves G., Mohammad S., Callender L.A., Coldewey S.M., Collino M., Greaves D.R, & Thiemermann C. (2020). X-Linked Immunodeficient Mice With No Functional Bruton's Tyrosine Kinase Are Protected From Sepsis-Induced Multiple Organ Failure. Frontiers in Immunology, 11, 581758.

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Quantification of Ascites Microparticle Subsets

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In a subgroup of 60 patients ascites MPs were stained with antibodies against surface antigens which allow to allocate their origin to either neutrophils (CD66b) or lymphocytes (CD3). For that purpose 20, non-survivors with low ascites MP levels (<488.4 MP/μL) were matched with two survivors, one with low and one with high ascites MP level, according to the MELD score and age. Respectively, 50 µL MPs were incubated with labeled antibodies FITC-CD3 (UCHT1, lymphocytes, Biolegend, San Diego, USA), APC-CD66b (G10F5; granulocytes, Biolegend, San Diego, USA) for 15 min at RT. 450 µL of cold sterile filtered (0.2 µm) FACS buffer (PBS, 1% BSA, 0.1% NaN₃) and 25 µL counting beads (Biolegend, San Diego, USA) were added. Analysis was performed on a FACS LSRFortessa using DiVa software (BD Bioscience, San Jose, USA) with the same approach and gates as described before. Prior measurements unbound antibody in FACS buffer as well as isotype controls (APC mouse IgM, FITC mouse IgG1; Biolegend, San Diego, USA) were run to exclude background and non-specific binding from real events. The number of positive MP was calculated relative to the number of all gated MPs by FACS (Additional file 1: Figure S1).

Engelmann C., Splith K., Krohn S., Herber A., Boehlig A., Boehm S., Pratschke J., Berg T, & Schmelzle M. (2017). Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study. Journal of Translational Medicine, 15, 188.

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