Calibrite apc beads

Manufactured by BD

Sourced in Germany

Calibrite APC beads are a type of fluorescent beads used for instrument calibration and quality control procedures in flow cytometry applications. They are designed to provide a consistent and reliable standard for the evaluation of instrument performance and fluorescence detection abilities.

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Lab products found in correlation

Anti cd4 clone gk1.5, by BD (3 mentions) Anti cd8 clone 53 6, by BD (3 mentions) Anti cd3e clone 145 2c11, by BioLegend (3 mentions) Anti cd45.2 clone 104, by BioLegend (3 mentions) Anti cd11b clone m1 70, by BioLegend (3 mentions) Lsr fortessa 2, by BD (2 mentions) Cytoflex s, by Beckman Coulter (2 mentions) Facscalibur, by BD (1 mentions) Cellquest software, by BD (1 mentions) Aria 3, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Mr frosty container, by Thermo Fisher Scientific (1 mentions) Vacuette k3edta tube, by Greiner (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Human fc block, by BD (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Cytofix cytoperm solution, by BD (1 mentions) Human serum albumin (hsa), by Gemini Bio (1 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions) Cd103, by BD (1 mentions)

Spelling variants of this product

Calibrite beads (59 citations) CaliBrite (11 citations) CaliBRITE APC calibration beads (2 citations)

7 protocols using calibrite apc beads

Quantification of Spinal Cord Immune Cells

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Single-cell suspensions from spinal cords were obtained via mechanical dissociation on a cell strainer. Immune cells were separated over a two-phase Percoll-density gradient. Staining of αβTCR/CD4⁺ T cells, αβTCR/CD8⁺ T cells and CD45/CD11b cells (macrophages/microglia) was performed using the following antibodies in a 1:200 dilution: Anti-CD3e (clone 145-2C11), BioLegend; anti-CD4 (clone GK 1.5), BD; anti-CD8 (clone 53-6.7), BD; anti-CD8 (clone 53–6.7), BD; anti-CD11b (clone M1/70), BioLegend; anti-CD45.2 (clone 104), BioLegend. The addition of Calibrite APC beads (BD) allowed for cell quantification. Flow cytometry was performed using a FACSCalibur operated by Cell Quest software (Becton Dickinson).

Berghoff S.A., Gerndt N., Winchenbach J., Stumpf S.K., Hosang L., Odoardi F., Ruhwedel T., Böhler C., Barrette B., Stassart R., Liebetanz D., Dibaj P., Möbius W., Edgar J.M, & Saher G. (2017). Dietary cholesterol promotes repair of demyelinated lesions in the adult brain. Nature Communications, 8, 14241.

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Lung Cell Isolation and Characterization

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The single-cell suspension (1/10 of the total lung) was liberated from erythrocytes using RCB buffer (ammonium chloride: 146 mmol, EDTA disodium: 0.2 mmol, sodium bicarbonate: 24 mmol). Antibodies were mixed in a solution of human immunoglobulins (c = 1.5 mg/ml, PZN-01700625, CSL Behring GmbH, Emil-von-Behring-Straße 76, 35041 Marburg, Germany) and 20 μl was added onto cell pellets, followed by a 20-min incubation at 4°C in the dark. Either 0.05 µg or 0.1 µg of each antibody were used per sample. Unbound antibodies were washed away, and the cell pellets were resuspended in PBS with 2 mM EDTA and 2% FCS (FACS buffer) for subsequent measurement on the BD LSR Fortessa II. Ten thousand Calibrite™ APC beads (BD Biosciences, Tullastraße 8-12, 69126 Heidelberg, Germany, 340487) were added to each sample to extrapolate the total cell number in the lung. BD Aria III was used for sorting AM for transcriptome and proteome analyses. Briefly, AM were identified as CD45⁺, CD11c⁺, and Siglec-F⁺ cells, whereas neutrophils were delineated based on CD45, CD11b, and Ly6G expression.

Zec K., Thiebes S., Bottek J., Siemes D., Spangenberg P., Trieu D.V., Kirstein N., Subramaniam N., Christ R., Klein D., Jendrossek V., Loose M., Wagenlehner F., Jablonska J., Bracht T., Sitek B., Budeus B., Klein-Hitpass L., Theegarten D., Shevchuk O, & Engel D.R. (2023). Comparative transcriptomic and proteomic signature of lung alveolar macrophages reveals the integrin CD11b as a regulatory hub during pneumococcal pneumonia infection. Frontiers in Immunology, 14, 1227191.

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PBMC Isolation and GRβ Receptor Analysis

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PBMCs were isolated from patient blood collected in a Vacuette K3 EDTA tube (Greiner Bio-One, Monroe, NC), using Histopaque-1077 (Sigma-Aldrich, St Louis, MO) density gradient centrifugation. Purified PBMCs were frozen at 5 × 106 cells per ml in cryopreservation media containing RPMI 1640, 11.25% human serum albumin (Gemini Bio-Products, West Sacramento, CA), and 10% DMSO (Sigma-Aldrich). Cells were stored in a Mr Frosty container (Thermo Fisher Scientific, Waltham, MA) at − 80 °C for 24 h before transferring to liquid nitrogen storage. Frozen human PBMCs were thawed, washed twice with RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 IU penicillin, and 100 μg/ml streptomycin, then rested overnight at 37 °C before staining. The following day, cells were treated with 2.5 μg Human Fc Block (BD Biosciences, San Jose, CA) for 20 min at room temperature. Samples were fixed and permeabilized for 20 min at 4 °C with 100 μl Cytofix/ Cytoperm solution (BD Biosciences). Cells were incubated with anti-human GRβ receptor polyclonal antibody (0.04 mg/ ml) for 20 min on ice, followed by a secondary goat anti-rabbit IgG APC (1:200; Thermo Fisher Scientific) for 20 min on ice. Thirty thousand cells were acquired on a BD FACS Aria II, standardized with Calibrite APC beads (BD Biosciences), and analyzed using FlowJo software (Treestar, Ashland, OR).

Whirledge S.D., Jewell C.M., Barber L.M., Xu X., Katen K.S., Garantziotis S, & Cidlowski J.A. (2017). Generating diversity in human glucocorticoid signaling through a racially diverse polymorphism in the beta isoform of the glucocorticoid receptor. Laboratory investigation; a journal of technical methods and pathology, 97(11), 1282-1295.

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Comprehensive Immune Cell Profiling

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We used the following monoclonal antibody clones: CD103 (PE, M290, BD); CD45 (APC-Cy7, APC and FITC, 30-F11, BD); F4/80 (PE-Cy7, BM8, BioLegend); CD11c (BV421 and BV605, N418, BioLegend); CD64 (PE, X54-5/7.1, BioLegend); MHC class II (APC and PE-Cy7, M5/114.15.2, BioLegend); Ly6C (PerCP-Cy5.5, AL-21, BD); and CD11b (APC and PE, M1/70, BD). Unspecific F_c receptor binding was blocked using human immune globulin, 10% liquid (Privigen) diluted 1:66. We determined absolute cell numbers by adding fixed numbers of CaliBRITE APC-beads (6 µm) (BD Biosciences) before measurement as internal reference, excluding dead cells with Hoechst-33258 (Invitrogen). Additionally, muscularis was weighed before digestion and cell numbers were normalised to 100 mg muscularis. Flow cytometry was performed on a BD LSRFortessa II (cytometer configuration is listed on https://cores.ukb.uni-bonn.de/fccf/wp-content/uploads/sites/11/2017/05/Filter-und-Fluorochrome-Fortessa-BUV.pdf) and data were analysed with Flow-Jo software (Tristar). Experiments including fluorescence activated cell sorting were performed on a BD Aria with a 70 µm nozzle (configuration: https://cores.ukb.uni-bonn.de/fccf/wp-content/uploads/sites/11/2017/02/Filter-und-Fluorochrome-Aria.pdf).

Pohl J.M., Gutweiler S., Thiebes S., Volke J.K., Klein-Hitpass L., Zwanziger D., Gunzer M., Jung S., Agace W.W., Kurts C, & Engel D.R. (2017). Irf4-dependent CD103+CD11b+ dendritic cells and the intestinal microbiome regulate monocyte and macrophage activation and intestinal peristalsis in postoperative ileus. Gut, 66(12), 2110-2120.

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Isolation and Analysis of Intrahepatic Leukocytes

Cited 1 time

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Flow cytometry was performed according to standard protocol.⁵ (link) The same amounts of liver tissue were digested by collagenase type IV (Roche Diagnostics GmbH, Mannheim, Germany) for 2 h and intrahepatic leukocytes were isolated through multiple differential centrifugation steps as previously published.¹⁴ (link) Samples were blocked with blocking buffer for 30 min. Immune cell extracts were stained with fluorochrome-conjugated antibodies of either a myeloid panel against CD11b (BV421; eBioscience, Frankfurt, Germany; 12-0112-82), CD45 (APC-Cy7; BD Biosciences, Erlangen, Germany; 557659), F4/80 (PE-Cy7; eBioscience, Frankfurt, Germany; 25-4801-82), Ly6G (AlexaFlour 700; Biolegend, San Diego, USA; 127622) or lymphoid panel against CD3 (APC; eBioscience, Frankfurt, Germany; 17-0031-82), CD4 (BV421; eBioscience, Frankfurt, Germany; 48-0041-82), CD8 (FITC; eBioscience, Frankfurt, Germany; 11-0081-85), CD19 (Alexa Flour 700; BD Biosciences, Erlangen, Germany; 551001), CD45 (APC-Cy7; BD Biosciences, Erlangen, Germany; 557659). Absolute cell numbers were determined by adding fixed numbers of Calibrite APC beads (BD Biosciences, Erlangen, Germany) as internal reference. Analysis was performed using FACS LSR Fortessa (BD Biosciences, Erlangen, Germany) and the acquired data was analyzed with FlowJo software (TreeStar, Ashland, OR).

Jaeger J.W., Brandt A., Gui W., Yergaliyev T., Hernández-Arriaga A., Muthu M.M., Edlund K., Elashy A., Molinaro A., Möckel D., Sarges J., Halibasic E., Trauner M., Kahles F., Rolle-Kampczyk U., Hengstler J., Schneider C.V., Lammers T., Marschall H.U., von Bergen M., Camarinha-Silva A., Bergheim I., Trautwein C, & Schneider K.M. (2024). Microbiota modulation by dietary oat beta-glucan prevents steatotic liver disease progression. JHEP Reports, 6(3), 100987.

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Isolation and Identification of Spinal Cord Immune Cells

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Single-cell suspensions from spinal cord were obtained via mechanical dissociation on a cell strainer. Immune cells were separated over a two-phase Percoll-density gradient by centrifugation. Staining of CD4+ T cells, CD8+ T cells and CD45/CD11b+ cells (macrophages/microglia) was performed using the following antibodies in a 1:200 dilution: anti-CD3e (clone 145-2C11, BioLegend), anti-CD4 (clone GK 1.5, BD), anti-CD8 (clone 53-6.7, BD), anti-CD11b (clone M1/70, BioLegend), anti-CD45.2 (clone 104, BioLegend).
The addition of CaliBRITE APC beads (BD) allowed for cell quantification. Flow cytometry was performed using a CytoFLEX S (Beckman Coulter) operated by CytExpert software (Beckman Coulter, v2.4).

Berghoff S.A., Spieth L., Sun T., Hosang L., Depp C., Sasmita A.O., Vasileva M.H., Scholz P., Zhao Y., Krueger-Burg D., Wichert S., Brown E.R., Michail K., Nave K.A., Bonn S., Odoardi F., Rossner M., Ischebeck T., Edgar J.M, & Saher G. (2021). Neuronal cholesterol synthesis is essential for repair of chronically demyelinated lesions in mice. Cell reports, 37(4).

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Isolation and Quantification of Spinal Cord Immune Cells

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Single-cell suspensions from spinal cord were obtained via mechanical dissociation on a cell strainer. Immune cells were separated over a two-phase Percoll-density gradient by centrifugation. Staining of CD4+ T cells, CD8+ T cells and CD45/CD11b+ cells (macrophages/microglia) was performed using the following antibodies in a 1:200 dilution: anti-CD3e (clone 145-2C11, BioLegend), anti-CD4 (clone GK 1.5, BD), anti-CD8 (clone 53-6.7, BD), anti-CD11b (clone M1/70, BioLegend), anti-CD45.2 (clone 104, BioLegend). The addition of CaliBRITE APC beads (BD) allowed for cell quantification. Flow cytometry was performed using a CytoFLEX S (Beckman Coulter) operated by CytExpert software (Beckman Coulter, v2.4) .

Berghoff S.A., Spieth L., Sun T., Hosang L., Depp C., Sasmita A.O., Vasileva M.H., Scholz P., Zhao Y., Krueger‐Burg D., Wichert S.P., Brown E.R., Michael K., Nave K., Bonn S., Odoardi F., Rossner M.J., Ischebeck T., Edgar J.M., & Saher G. (2021). Neuronal cholesterol synthesis is essential for repair of chronically demyelinated lesions in mice.

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