Calibur

Manufactured by BD

Sourced in United States, Germany

Calibur is a compact, high-performance flow cytometer designed for routine laboratory analysis. It provides accurate and reliable measurements of various cellular parameters, including size, granularity, and fluorescence intensity. Calibur is equipped with advanced optics and fluidics systems to ensure consistent and reproducible results.

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Close spelling variants of this product

BD FACS Calibur flow cytomyter (4 citations) FACS Calibur 3CB (3 citations) FACS Calibur-S System (8 citations) Calibur Becton Dickinson flow cytometer (4 citations) Calibur system (6 citations) FASC Calibur MT flow cytometer (6 citations) BD FACS-Calibur Cell Cytometer (2 citations) BD FACS Calibur flow cytometer instrument (2 citations) BD FACSCalibur Calibur Flow Cytometer 4 Color (2 citations) FACS Calibur/LSRII flow cytometer (4 citations)

250 protocols using calibur

Fluorescent Antibody Cell Sorting Protocols

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The cells were labeled with the fluorescent antibodies as described in
details elsewhere. [63 (link), 71 –72 (link),
79 ] They were sorted on the Calibur,
Vantage SE, or Aria (Becton-Dickinson). The antibodies were dissolved and all
washing steps carried in phenol-free, Ca+ / Mg+- free, PIPES buffered saline
solution, supplemented with 20 mM glucose, 10% human serum. Sorting was
performed on Aria, Calibur, Vantage SE (Becton-Dickinson) with the sheath
pressure set at 20 pounds per square inch and low count rate. The sorted batches
were analyzed on Calibur or Aria using FACS Diva software or on the FC500
(Beckman-Coulter). For the measurement of the fluorescently labeled cells, these
settings were tuned at the maximum emission for the Eu chelated antibody at 500V
with references to isotype antibodies and non-labeled cells. This assured the
comparisons between populations of cells labeled with multiple antibodies
without changing the settings on PMTs.
The fluorescently labeled cells or tissues were imaged with the Axiovert
(Zeiss) equipped with the Enterprise argon ion (457 nm, 488 nm, 529 nm lines)
and ultraviolet (UV) (364 nm line) lasers; Odyssey XL digital high-sensitivity
with instant deconvolution confocal laser scanning imaging system operated up to
240 frames/s (Noran), and the Diaphot (Nikon) equipped with the
diode-pumped Nd:YLF solid state laser (1048 nm line) (Microlase).

Malecki M, & Saetre B. (2018). HIV Apheresis Tags (HIVAT) Aided Elimination of Viremia. Molecular and cellular therapies, 6(1), 6.

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Multicolor Fluorescence-Activated Cell Sorting

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The cells were labeled with the fluorescent antibodies as described in
details elsewhere. [25 (link)–26 (link), 29 –30 ] They were
sorted on the Calibur, Vantage SE, or Aria (Becton-Dickinson). The antibodies
were dissolved and all washing steps carried in phenol-free, Ca+ / Mg+- free,
PIPES buffered saline solution, supplemented with 20 mM glucose, 10% human
serum. Sorting was performed on Aria, Calibur, Vantage SE (Becton-Dickinson)
with the sheath pressure set at 20 pounds per square inch and low count rate.
The sorted batches were analyzed on Calibur or Aria using FACS Diva software or
on the FC500 (Beckman-Coulter). For the measurement of the fluorescently labeled
cells, these settings were tuned at the maximum emission for the Eu chelated
antibody at 500V with references to isotype antibodies and non-labeled cells.
This assured the comparisons between populations of cells labeled with multiple
antibodies without changing the settings on PMTs.
The fluorescently labeled cells or tissues were imaged with the Axiovert
(Zeiss) equipped with the Enterprise argon ion (457 nm, 488 nm, 529 nm lines)
and ultraviolet (UV) (364 nm line) lasers; Odyssey XL digital high-sensitivity
with instant deconvolution confocal laser scanning imaging system operated up to
240 frames/s (Noran), and the Diaphot (Nikon) equipped with the
diode-pumped Nd:YLF solid state laser (1048 nm line) (Microlase).

Malecki M, & Saetre B. (2018). HIV Universal Vaccine. Molecular and cellular therapies, 6(1), 5.

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Quantifying EGFP, mCherry, and Apoptosis

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For quantifying the ectopic EGFP and mCherry expression in cells, cells were collected by trypsinization at 24 h post transfection and quantified with flow cytometry (Calibur, BD, USA). For quantifying cell apoptosis, cells were collected by trypsinization at 24 h post transfection and detected with the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BD, USA) according to the manufacturer’s instructions. The fluorescence intensity of cells was quantified with flow cytometry (Calibur, BD, USA). Flow cytometry data analysis and figure preparation were done using BD software.

Gao J., Luo T., Lin N., Zhang S, & Wang J. (2020). A New Tool for CRISPR-Cas13a-Based Cancer Gene Therapy. Molecular Therapy Oncolytics, 19, 79-92.

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SIRPα Binding Assay in AML Cells

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SIRPα binding was studied using THP-1 and U937 AML cell lines. The cells were incubated for 0.5 h at 4°C with increasing concentrations of BR105. The cells were then stained with FITC-labeled goat anti-human IgG conjugates (Abcam), and analyzed by flow cytometry (Calibur, BD Biosciences).
For binding to CD14+ monocytes, macrophages and neutrophils, cells were blocked with human Fc receptor Blocking Reagent (BD Biosciences) for 20 min at room temperature. The cells were then incubated for 0.5 h at 4°C with 10 µg/mL BR105. Bound antibodies were detected using Goat anti-Human IgG F(ab')₂-FITC conjugates (Invitrogen), and cells were analyzed by flow cytometry (Calibur, BD Biosciences).

Wu Z.H., Li N., Mei X.F., Chen J., Wang X.Z., Guo T.T., Chen G., Nie L., Chen Y., Jiang M.Z., Wang J.T, & Wang H.B. (2022). Preclinical characterization of the novel anti-SIRPα antibody BR105 that targets the myeloid immune checkpoint. Journal for Immunotherapy of Cancer, 10(3), e004054.

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Cell Cycle and Apoptosis Analysis

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Cell-cycle analysis: Pre-cooled 75% ethanol was used to fix PCa cells (1 × 10⁶ cells) at 4 °C overnight. Subsequently, the cells were washed with PBS three times and resuspended with 500 µl propidium iodide (PI) (Becton, Dickinson and Company, USA) at room temperature. After 15 min, the cells were loaded into the flow cytometer (Calibur, BD Bioscience, USA) for analysis. FlowJo software (Tree Star, San Carlos, USA) was used to evaluate the results.
Cell apoptosis analysis: The PCa cells (1 × 10⁶ cells) were washed with pre-cooled PBS twice and resuspended in 1 ml of 1× binding buffer. Then, 5 µl FITC Annexin V and 5 µl PI (Becton, Dickinson and Company) were added to 100 µl of cell suspension (1 × 10⁵ cells), and the cells were incubated for 15 min at room temperature. Finally, 200 µl of 1× binding buffer was added to each tube, and cell apoptosis analysis was detected via a flow cytometer (Calibur, BD Bioscience). FlowJo software (Tree Star) was used to analyze the results.

Lai W., Zhu W., Xiao C., Li X., Wang Y., Han Y., Zheng J., Li Y., Li M, & Wen X. (2021). HJURP promotes proliferation in prostate cancer cells through increasing CDKN1A degradation via the GSK3β/JNK signaling pathway. Cell Death & Disease, 12(6), 583.

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Identifying antigen-specific CD8+ T cells

Cited 1 time

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Single-cell suspensions were first pretreated with anti-CD16/32 (2.4G2) antibodies to block Fc receptors and then stained with anti-CD11c (HL3), anti-MHC class II (M5/114.15.2), anti-CD317 (927), anti-Siglec-H (551), or anti-CD45.2 (104) antibodies. Live cells were gated based on 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) exclusion. Multiple cell populations were separated by flow cytometry (LSR Fortessa or Calibur; BD Biosciences). H-2D_b tetramers specific for the RSV M_187–195 peptide (NAITNAKII) were prepared with streptavidin-allophycocyanin (APC), using the protocol of the NIH Tetramer Core Facility (https://tetramer.yerkes.emory.edu/support/protocols) [17 (link)]. Single-cell suspensions from the lungs of immunized mice were pretreated with anti-CD16/32 (2.4G2) antibodies to block Fc receptors and then stained with anti-CD8α (53-6.7) and anti-CD3e (145-2C11) antibodies. Then, APC-labelled tetramer staining was performed. The number of RSV M_187–195 peptide-specific CD8⁺ T cells was analyzed by flow cytometry (Calibur; BD Bioscience). Final analysis and graphical output of the results were obtained using FlowJo software (Version 9, Tree Star, Inc., Ashland, OR, USA).

Kim T.H., Oh D.S., Jung H.E., Chang J, & Lee H.K. (2019). Plasmacytoid Dendritic Cells Contribute to the Production of IFN-β via TLR7-MyD88-Dependent Pathway and CTL Priming during Respiratory Syncytial Virus Infection. Viruses, 11(8), 730.

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CD133 Expression and Cell Cycle Analysis

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Cells were fixed on ice for 20 min with 2% paraformaldehyde, blocked with 10% NGS for 1 h, followed by primary antibody incubation with PE-conjugated CD133 (Miltenyi) for 30 min. Labeled cells were analyzed using a BD Calibur, with unlabeled cells serving as negative controls. For cell cycle analysis, cells were pre-treated for 24 h with 100 ng/mL Sema3A, dissociated, and fixed with cold 70% ethanol for 30 min at 4 ^oC. Cells were washed and treated with RNAse (Qiagen). Propidium Iodide (PI) was added to the cells and analysis was conducted using a BD Calibur, with post-analysis using FlowJo software version 7.6.5.

Higgins D.M., Caliva M., Schroeder M., Carlson B., Upadhyayula P.S., Milligan B.D., Cheshier S.H., Weissman I.L., Sarkaria J.N., Meyer F.B, & Henley J.R. (2020). Semaphorin 3A mediated brain tumor stem cell proliferation and invasion in EGFRviii mutant gliomas. BMC Cancer, 20, 1213.

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In Vitro Mesenchymal Stem Cell Migration Assay

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The in vitro migration assay was performed in the transwell inserts, with an 8-μm pore size, the membrane of which was uncoated (SPL, Germany). The cells were treated by PBS (Phosphate buffered saline) with 10-100 or 500 nM kp-10 (Anaspec, USA) for 24 hours and one other group of cells was not treated with kp-10. Then the cells were trypsinized and a number of 2 × 10⁴ mesenchymal stem cells in 200 μl serum-free medium were seeded into the top chamber. Then, 30% of Fetal calf serum (FCS) or 4 × 10⁴ melanoma cells in 500 μl medium were added to the lower chamber as a chemoattractant. After 24 hours of incubation at 37˚C, the non-migrated cells were removed from the top of the membrane, whereas, cells on the bottom of the membrane were trypsinized with 300 μl of trypsin and were suspended into the medium and counted in five minutes by flow cytometry (BD Calibur^™ (Becton Dickinson)) after obtaining the appropriate gate. Results were obtained by three experiments.[23 (link)24 (link)]

Golzar F., Javanmard S.H., Bahrambeigi V, & Rafiee L. (2015). The effect of Kisspeptin-10 on mesenchymal stem cells migration in vitro and in vivo. Advanced Biomedical Research, 4, 20.

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Cell Cycle Analysis by Flow Cytometry

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TE1 cell cycle distribution was measured using a flow cytometer by quantifying the PI labeled DNA content. After transfection with recombinant plasmids for 48 h, TE1 cells were collected and fixed with 70% cold ethanol at 4°C for 30 min, then washed with PBS. Pellets were re-suspended and incubated in PBS containing RNaseA (50 µg/ml), Triton (0.2%) and PI (20 µg/ml) at 4°C for 30 min in the dark. Cell cycle analysis was performed using a flow cytometer (BD Calibur; Becton-Dickinson and Company). Data were analyzed with Cell Quest data acquisition and analysis software (Becton-Dickinson and Company) after 1 h.

Zhou C., Ma J., Lu Y., Zhao W., Xu B., Lin J., Ma Y., Tian Y., Zhang Q., Wang W., Yan W, & Jiao P. (2020). TERT promoter regulating melittin expression induces apoptosis and G0/G1 cell cycle arrest in esophageal carcinoma cells. Oncology Letters, 21(1), 16.

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Characterizing OC Stem Cell Markers

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Primary isolated OC were cultured in vitro and passaged for about 6-8 times. The OCs were harvested and then incubated with anti-EpCAM-APC, anti-Sca-1-PE, anti-CD44-PE, anti-CD49f-APC, anti-CD45-APC, anti-CD34-PE, anti-c-kit-FITC, anti-α6 integrin-APC, anti-β1 integrin-PercpCy5.5, anti-β4 integrin-PercpCy5.5 (all from ebioscience, San Diego, CA) at 4°C for 30 mins, after that, cells were fixed with 1% paraformaldehyde and analyzed by a BD Calibur (Becton, Dickinson and Company, New Jersey) and Flowjo software (Tree Star, San Carlos, CA).

Qian L., Zhang H., Gu Y., Li D., He S., Wang H., Cheng Y., Yang W., Yu H., Zhao X., Cai W., Meng L., Jin M., Wang Y, & Zhang Y. (2018). Reduced production of laminin by hepatic stellate cells contributes to impairment in oval cell response to liver injury in aged mice. Aging (Albany NY), 10(12), 3713-3735.

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