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Count beads

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Count beads are small, spherical particles used in various laboratory applications to assist in the accurate counting and quantification of cells, particles, or other analytes. They serve as a reference standard to calibrate and validate cell counting and flow cytometry instruments.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (4 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Cytek Biosciences (2 mentions) Celltrace yellow, by Thermo Fisher Scientific (2 mentions) Ccl26, by Thermo Fisher Scientific (2 mentions) Facs lysis buffer, by BD (1 mentions) Gentlemacs c tube, by Miltenyi Biotec (1 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions) Collagenase type 4, by Worthington (1 mentions) Dnase 1, by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Golgistop, by BD (1 mentions)

6 protocols using count beads

1

Cytotoxicity Assay for Leukemia Cells

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Target leukemia cells were stained with CFSE (Tonbo) or CellTrace Violet (Thermo) and T cells were left unstained or stained with CellTrace Yellow prior to coculture. Target cells and T cells were cocultured in 96 well u-bottom plates for up to 72 h. Count beads (Invitrogen) were added to each sample to determine absolute cell counts. After coculture, cells were stained with Near IR Live/Dead for viability and fixed with 4% formaldehyde prior to flow cytometric analysis. Test compounds for which multiple donors or multiple replicates were unobtained, or T cell donors from whom drug-induced lysis was not observed, were excluded from analysis. Specific lysis was calculated using the equation: %specific lysis = 100(%treated sample [violet+NIR LD+] - %isobeads[violet+NIR LD+])/(100–%isobeads[violet +NIR LD+]). Lysis measurements in all cases, including blinatumomab-treated controls, were <100%.
Do P., Perdue L.A., Chyong A., Hunter R., Dougan J., Henry C.J., Porter C.C, & Dreaden E.C. (2020). Rapid Assembly and Screening of Multivalent Immune Cell-Redirecting Therapies for Leukemia. ACS combinatorial science, 22(10), 533-541.
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2

Quantifying Lung Immune Cells post Influenza

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On day 7 post influenza virus infection, mice were euthanized and lungs were homogenized in GentleMACS C tubes (Miltenyi) containing Dnase I (Sigma Aldrich) and type IV collagenase (Worthington Biochemical Corporation). Lungs were processed into single cell suspensions using GentleMACS equipment. Single cell homogenates were blocked in CellBlox (Invitrogen) and viability stain was performed using eFluor 780 Fixable Viability Dye (Invitrogen) according to the manufacturer’s instructions. Cells were washed and stained with Invitrogen antibodies for CD45, Ly6G and Ly6C (catalogue number M001T02R02, 46-9668-82, and 48-5932-82, respectively) for 30 minutes on ice. Cells were fixed with BD FACS lysis buffer for 10 minutes at 4°C, resuspended in PBS, and run on a Cytek Aurora instrument. Count beads (Invitrogen) were added to each sample for quantification of total cell number. Samples were analyzed using FlowJo Software (10.8.1).
Speaks S., Zani A., Solstad A., Kenney A., McFadden M.I., Zhang L., Eddy A.C., Amer A.O., Robinson R., Cai C., Ma J., Hemann E.A., Forero A, & Yount J.S. (2023). Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation. bioRxiv.
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3

Eosinophil Migration Assay

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Following overnight culture with- and without IL-5, 2×104 eosinophils were resuspended in coculture media and seeded into well inserts with 5.0-μm pore size and increasing concentrations of EPC2-conditioned media or 5 ng/mL CCL26 (Preprotech cat no. 300-48) in coculture media were added to lower wells. Cells were incubated at 37°C for 4 h, at which time all cells in lower wells were collected and fixed. Using count beads (Invitrogen cat no. C36950), total cell counts were enumerated by flow cytometry. Percent of migrated cells in response to chemokine were normalized to the number of cells recovered from the lower well when no chemokine was present.
Dunn J.L., Caldwell J.M., Ballaban A., Ben-Baruch Morgenstern N., Rochman M, & Rothenberg M.E. (2021). Bidirectional Crosstalk Between Eosinophils and Esophageal Epithelial Cells Regulates Inflammatory and Remodeling Processes. Mucosal immunology, 14(5), 1133-1143.
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4

Intracellular Cytokine Analysis of ILC2

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After 6 days in culture, ILC2 were collected and restimulated with 50ng/ml PMA (Sigma-Aldrich), 1µg/ml ionomycin (Sigma-Aldrich), and 0.07% golgi stop (BD Biosciences) for 4 hours at 37°C. ILC2 were then surface stained, fixed and permeabolized using methods described in above section. ILC2 were then intracellularly stained with PE anti-gata3 (clone TWAJ), APC anti-IL-5 (clone TRFK5) and eFluor 450 anti-IL-13 (clone eBio13A). Just prior to flow cytometry analysis, count beads (Invitrogen) were added in per manufacturer’s protocol to determine total ILC2 number.
Cephus J., Stier M., Fuseini H., Yung J., Toki S., Bloodworth M., Zhou W., Goleniewska K., Zhang J., Garon S., Hamilton R., Poloshukin V., Boyd K., Peebles R J.r, & Newcomb D. (2017). Testosterone attenuates group 2 innate lymphoid cell-mediated airway inflammation. Cell reports, 21(9), 2487-2499.
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5

Cytotoxicity Assay for Leukemia Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Target leukemia cells were stained with CFSE (Tonbo) or CellTrace Violet (Thermo) and T cells were left unstained or stained with CellTrace Yellow prior to coculture. Target cells and T cells were cocultured in 96 well u-bottom plates for up to 72 h. Count beads (Invitrogen) were added to each sample to determine absolute cell counts. After coculture, cells were stained with Near IR Live/Dead for viability and fixed with 4% formaldehyde prior to flow cytometric analysis. Test compounds for which multiple donors or multiple replicates were unobtained, or T cell donors from whom drug-induced lysis was not observed, were excluded from analysis. Specific lysis was calculated using the equation: %specific lysis = 100(%treated sample [violet+NIR LD+] - %isobeads[violet+NIR LD+])/(100–%isobeads[violet +NIR LD+]). Lysis measurements in all cases, including blinatumomab-treated controls, were <100%.
Do P., Perdue L.A., Chyong A., Hunter R., Dougan J., Henry C.J., Porter C.C, & Dreaden E.C. (2020). Rapid Assembly and Screening of Multivalent Immune Cell-Redirecting Therapies for Leukemia. ACS combinatorial science, 22(10), 533-541.
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6

Eosinophil Migration Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Following overnight culture with- and without IL-5, 2×104 eosinophils were resuspended in coculture media and seeded into well inserts with 5.0-μm pore size and increasing concentrations of EPC2-conditioned media or 5 ng/mL CCL26 (Preprotech cat no. 300-48) in coculture media were added to lower wells. Cells were incubated at 37°C for 4 h, at which time all cells in lower wells were collected and fixed. Using count beads (Invitrogen cat no. C36950), total cell counts were enumerated by flow cytometry. Percent of migrated cells in response to chemokine were normalized to the number of cells recovered from the lower well when no chemokine was present.
Dunn J.L., Caldwell J.M., Ballaban A., Ben-Baruch Morgenstern N., Rochman M, & Rothenberg M.E. (2021). Bidirectional Crosstalk Between Eosinophils and Esophageal Epithelial Cells Regulates Inflammatory and Remodeling Processes. Mucosal immunology, 14(5), 1133-1143.
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