Countbright

Manufactured by Thermo Fisher Scientific

Sourced in United States

CountBright is a compact and automated cell counter designed for accurate cell enumeration. The device utilizes advanced optics and image processing technology to provide reliable cell counts across a wide range of sample types. Its intuitive interface and streamlined workflow enable efficient cell quantification in a laboratory setting.

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Lab products found in correlation

Facscanto 2, by BD (6 mentions) Facsaria, by BD (3 mentions) Dnase 1 collagenase, by Merck Group (2 mentions) Cd8 microbead, by Miltenyi Biotec (2 mentions) Facs canto flow cytometer, by Thermo Fisher Scientific (2 mentions) Diva software, by BD (2 mentions) Cd11b, by BioLegend (2 mentions) Megamix, by Stago (2 mentions) Flow count, by Beckman Coulter (1 mentions) Trucount, by BD (1 mentions) Cell counting kit 8 cck 8 assay kit, by Beyotime (1 mentions) Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions) Mitoxantrone, by Merck Group (1 mentions) Fc block, by BioLegend (1 mentions) Facscanto flow cytometer, by BD (1 mentions) Ultracomp ebeads, by Thermo Fisher Scientific (1 mentions) Facsdiva software, by BD (1 mentions) Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions) Lsr 2 fortessa cytometer, by BD (1 mentions) Anti his apc, by R&D Systems (1 mentions)

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39 protocols using countbright

Comparative Analysis of Counting Beads

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Three different popular brands of counting beads were compared: TruCount (BD Biosciences; size not specified), Flow-Count (Beckman Coulter; 10 μm diameter) and CountBright (Molecular Probes, Eugene, Oregon, USA; 7 μm diameter). Each bead preparation was diluted into the final test volume at the indicated concentration by adding exact quantities based on the individual lot concentration provided by the manufacturer. The plasma source, concentration and acquiring time on the flow cytometer were kept constant. For experiments comparing FSC with SSC, CountBright beads were used at a final concentration of 50 beads/μl.

Alkhatatbeh M.J., Enjeti A.K., Baqar S., Ekinci E.I., Liu D., Thorne R.F, & Lincz L.F. (2018). Strategies for enumeration of circulating microvesicles on a conventional flow cytometer: Counting beads and scatter parameters. Journal of Circulating Biomarkers, 7, 1849454418766966.

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Cell Proliferation Measurement Techniques

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Cell proliferation was assessed as following: (1) Cell Counting Kit-8 (CCK-8) assay kit (Beyotime, China): Briefly, cells were seeded in a 96-well cell culture cluster plates, at a density of 2×10⁴ cells per well in 100μL of culture medium. Then 10μL of CCK-8 reagents were added to each well and incubated for 2 hours at 37°C, and the absorbance of the samples was measured at 450nm using an enzyme-linked immunosorbent assay plate reader. (2) Cell cycle analysis: Cells were fixed in 70% ethanol in PBS at -20°C overnight, and then resuspended in PBS containing 40μg/mL PI and 100μg/mL RNAse and incubated for 30 min at 37°C in darkness. Samples were analyzed by flow cytometry. (3) Cell count using the CountBright (Molecular Probes, Eugene, USA): 300μl of cells was stained and 50μl of CountBright absolute counting beads was added. Then cell count was analyzed by flow cytometry.

Fang J., Huang X., Han X., Zheng Z., Hu C., Chen T., Yang X., Ouyang X., Chen Z, & Wei H. (2020). Endothelial progenitor cells promote viability and nerve regenerative ability of mesenchymal stem cells through PDGF-BB/PDGFR-β signaling. Aging (Albany NY), 12(1), 106-121.

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AML Blasts Coculture with Mesenchymal Stromal Cells

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Primary AML mononuclear cells were plated at 2×10⁵/cm² in SynH (Abcell-Bio) supplemented with L-Glutamine, non-essential amino acids and 10% fetal bovine serum for 3-4 days on confluent MSC isolated from the BM of either AML patients (n=9) or HD (n=5) (Online Supplementary Appendix). AML blasts were cultured without MSC feeders for controls. Transwell and neutralization experiments are described in the Online Supplementary Appendix.
In some experiments, mitoxantrone (50nM; Sigma Aldrich) was added to the co-culture for 24 hours (h) and cells were pretreated or not with verapamil (50 mM) for 2 h before adding mitoxantrone and during mitoxantrone treatment.
At the end of co-cultures, non-adherent cells were flushed and stained with anti-CD45 antibody and annexin V (Invitrogen). Counting beads (CountBright, Life Technologies) were added to cell suspension to quantify cell populations by flow cytometry using Fortessa apparatus with Diva software (Becton Dickinson).

Boutin L., Arnautou P., Trignol A., Ségot A., Farge T., Desterke C., Soave S., Clay D., Raffoux E., Sarry J.E., Malfuson J.V., Lataillade J.J., Le Bousse-Kerdilès M.C, & Anginot A. (2020). Mesenchymal stromal cells confer chemoresistance to myeloid leukemia blasts through Side Population functionality and ABC transporter activation. Haematologica, 105(4), 987-9998.

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Hif1an Knockout Immune Cell Analysis

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Female Hif1an^fl/fldLck Cre- (‘WT’) and Hif1an^fl/fldLck Cre+ (‘FIH KO’) mice, aged between 7 - 12 weeks, were sacrificed and lymph nodes, spleen and liver were harvested. Single cell suspensions were created by passing each organ through a 40 μM strainer (VWR). Cells were incubated with Fc block (Biolegend) followed by surface antibody staining, as outlined previously. Samples were analysed using a FACSCanto II flow cytometer (BD Biosciences) and counting beads were used to quantify absolute cell numbers (CountBright, Life Technologies).

Bargiela D., Cunha P.P., Veliça P., Krause L.C., Brice M., Barbieri L., Gojkovic M., Foskolou I.P., Rundqvist H, & Johnson R.S. (2024). The factor inhibiting HIF regulates T cell differentiation and anti-tumour efficacy. Frontiers in Immunology, 15, 1293723.

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Cell Viability Determination Protocols

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Cells were either counted using an automated cell counter (BioRad TC20) with viability assessed by trypan blue exclusion or were stained with LIVE/DEAD (Life Technologies) viability dye and absolute numbers were determined by flow cytometry using counting beads (Countbright, Life Technologies).

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Isolation and Characterization of Murine Lung Cells

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BAL cells were obtained after centrifugation of pooled BAL fluid, and the cell pellet was stained with fluorochrome-labelled antibodies to obtain a leukocyte differential as in Abdulnour et al.^{36 (link)}; neutrophils (CD45⁺ F4/80⁻ Ly6G⁺ CD11b⁺), infiltrating macrophages (iMacs; CD45⁺ SSc^High F4/80⁺ CD11c^Low CD11b⁺ SiglecF–Ly6C+), exudative macrophages (ExMacs; CD45⁺ SSc^High F4/80⁺ CD11c^High CD11b⁺ SiglecF+ Ly6C+), and resident alveolar macrophages (rAM; CD45⁺ F4/80⁺ SSc^High CD11c^High CD11b⁻ SiglecF+ Ly6C−). Cell counts were obtained by adding a known amount of fluorescent counting beads (CountBright; Life Technologies) to the BAL cell pellet prior to flow cytometry as per the manufacturer’s recommendations, and normalized to lavage. FACSCanto II (BD Biosciences, San Jose, CA, USA) and FlowJo Ver. 10 software (Tree Star, Ashland, OR) were used for analyses. In some experiments, whole murine lungs were homogenized 24 h after ALI and a cell suspension was prepared as in.^{31 (link)} Neutrophils (CD45+ Ly6G+), macrophages (CD45+ SSc^High F4/80+), lung epithelial cells (CD45− CD31− CD326+), and lung endothelial cells (CD45− CD31+) were sorted with >95% purity, using FACSAria (BD Biosciences, San Jose, CA, USA). The sorted populations were used for RNA extraction and RT-PCR.

Abdulnour R.E., Howrylak J.A., Tavares A.H., Douda D.N., Henkels K.M., Miller T.E., Fredenburgh L.E., Baron R.M., Gomez-Cambronero J, & Levy B.D. (2018). Phospholipase D isoforms differentially regulate leukocyte responses to acute lung injury. Journal of leukocyte biology, 103(5), 919-932.

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Quantifying Osx/GFP+ Cells in T-ALL

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T-ALL engraftment and infiltration was confirmed via peripheral blood sampling and/or tibia puncture. Once mice presented with signs of ill health (as described earlier), mice were euthanized and bones digested with a DNAse I/Collagenase (Sigma) solution. The total number of Osx/GFP⁺ cells was assessed by flow cytometry analysis using counting beads (CountBright, Life Technologies).

Hawkins E.D., Duarte D., Akinduro O., Khorshed R.A., Passaro D., Nowicka M., Straszkowski L., Scott M.K., Rothery S., Ruivo N., Foster K., Waibel M., Johnstone R.W., Harrison S.J., Westerman D.A., Quach H., Gribben J., Robinson M.D., Purton L.E., Bonnet D, & Lo Celso C. (2016). T cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments. Nature, 538(7626), 518-522.

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Characterization of Lung Macrophage Subsets

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BAL cells were obtained after centrifugation of pooled BAL fluid, and the cell pellet was stained with fluorochrome-labelled antibodies to obtain a leukocyte differential; neutrophils (CD45⁺ F4/80⁻ Ly6G⁺ CD11b⁺), infiltrating macrophages (iMacs; CD45⁺ SSc^High F4/80⁺ CD11c^Low CD11b⁺), exudative macrophages (ExMacs; CD45⁺ SSc^High F4/80⁺ CD11c^High CD11b⁺), and resident alveolar macrophages (rAM; CD45⁺ F4/80⁺ SSc^High CD11c^High CD11b⁻). Cell counts were obtained by adding a known amount of fluorescent counting beads (CountBright, Life Technologies) to the BAL cell pellet prior to flow cytometry as per the manufacturer’s recommendations, and normalized to lavage. FACSCanto II (BD Biosciences) and FlowJo Ver. 10 software (Tree Star, Ashland, OR, USA) were used for analyses. In some experiments, five BALFs were pooled and F4/80 positive macrophage subsets cells were sorted with > 95% purity, using FACSaria (Beckton-Dickinson). The sorted populations were used for ex vivo efferocytosis assays. For sorting of macrophage subsets, BALF obtained 72 hrs after intra-bronchial LPS was used, as LPS resulted in the highest maximal number of macrophages (see Results) allowing for a better yield of each macrophage subset using fluorescence activated cell sorting.

Abdulnour R.E., Sham H.P., Douda D.N., Colas R.A., Dalli J., Bai Y., Ai X., Serhan C.N, & Levy B.D. (2015). Aspirin-triggered resolvin D1 is produced during self-resolving gram-negative bacterial pneumonia and regulates host immune responses for the resolution of lung inflammation. Mucosal immunology, 9(5), 1278-1287.

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Adoptive Transfer of OT-I T Cells in Nitric Oxide Synthase Knockout Mice

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OT-I T cells purified with CD8⁺ microbeads (Miltenyi Biotec) from spleens of Nos2^fl/fl (WT; CD45.2) and Nos2^fl/fldlck^CRE (NOS2^KO; CD45.1/CD45.2) C57BL/6j animals. WT (control) and NOS2^KO cells were then mixed 1:1, and a total of 2 million cells (1 million of each genotype) was injected intraperitoneally into C57BL/6J CD45.1⁺ WT host mice. Endogenous and adoptive populations were distinguished by the allelic variants of CD45. One day later, host mice were vaccinated intraperitoneally with 8×10⁵ SIINFEKL-loaded BMDMs. Peripheral blood was collected from the tail vein at days 7 and 10 after T-cell transfer and analyzed by flow cytometry following antibody staining and treatment with BD FACS lysing solution, as specified below. On day 30, animals were reinjected with 8×10⁵ SIINFEKL-loaded BMDMs, and 7 days later, the spleens, inguinal lymph nodes, and liver tissues were harvested, mashed in 40-μm cell strainers into single-cell suspensions as previously described, and analyzed by flow cytometry to determine recall responses. Animals injected with PBS on day 30 were used as negative controls of the recall response. Animals were assigned randomly to each experimental group. Absolute numbers of adoptively transferred CD8⁺ OT-I cells were determined with the use of counting beads (CountBright, Life Technologies).

Cunha P.P., Bargiela D., Minogue E., Krause L.C., Barbieri L., Brombach C., Gojkovic M., Marklund E., Pietsch S., Foskolou I., Branco C.M., Veliça P, & Johnson R.S. (2022). Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis. Cancer Immunology Research, 11(3), 351-363.

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Neutrophil Identification in BALF

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Following the collection of BALF and removal of supernatant, cells were stained with CD45, CD11B, CD11C, and Ly6G (BioLegend, San Diego, CA). Neutrophils were identified as (CD45⁺, CD11B⁺, CD11C⁻ and Ly6G⁺). Cell counts were obtained by adding a known amount of fluorescent counting beads (CountBright, Life Technologies, Carlsbad, CA) to the BALF cell pellet prior to flow cytometry as per the manufacturer’s recommendation, and normalized to lavage. FACSCanto II (BD Biosciences, San Jose, CA) and FlowJo Ver. 10 software (Tree Star, Ashland, OR) were used for analyses.

Sham H.P., Walker K.H., Abdulnour R.E., Krishnamoorthy N., Douda D.N., Norris P.C., Barkas I., Benito-Figueroa S., Colby J.K., Serhan C.N, & Levy B.D. (2018). 15-epi-Lipoxin A4, Resolvin D2 and Resolvin D3 induce NF- κB regulators in bacterial pneumonia. Journal of immunology (Baltimore, Md. : 1950), 200(8), 2757-2766.

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