Anti cd45 krome orange

Manufactured by Beckman Coulter

Sourced in France, United States

The Anti-CD45-Krome orange is a fluorochrome-conjugated antibody designed for the detection and identification of CD45-positive cells using flow cytometry. It provides a specific and sensitive tool for the analysis of various hematopoietic cell populations.

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Lab products found in correlation

Navios, by Beckman Coulter (2 mentions) Anti cxcr4 pe, by Beckman Coulter (1 mentions) Anti cd3 pacific blue, by BD (1 mentions) Anti cd14 apc h7, by BD (1 mentions) Paraformaldehyde pfa, by Bio-Rad (1 mentions) Facsuite software, by BD (1 mentions) Anti cd8 pe, by BD (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Anti cd4 fitc, by BD (1 mentions) Facslyric, by BD (1 mentions) Anti cd56 apc, by BD (1 mentions) Anti cd3 percp cy5, by BD (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Intraprep permeabilization reagent, by Beckman Coulter (1 mentions) Calcium ionophore, by Merck Group (1 mentions) Pe anti cd45ro, by BioLegend (1 mentions) Anti cd3 apc, by Beckman Coulter (1 mentions) Anti cd4 apc alexafluor750, by Beckman Coulter (1 mentions) Anti il4 pe cy7, by BioLegend (1 mentions) Lsr 2, by BD (1 mentions)

6 protocols using anti cd45 krome orange

Fibrocyte Characterization and Identification

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After 8 days of culture (T8), adherent fibrocytes were lifted by incubation in ice-cold 0.05% EDTA in phosphate-buffered saline (PBS), and cell viability was determined by the trypan blue exclusion test.
Characterization and identification of fibrocytes was performed at T0 and T8 by FACS (Beckman Coulter Company) using anti-CD45 (anti-CD45-krome orange, Beckman Coulter Company), anti-COL I (anti-COL I-FITC, Milli-Mark, Millipore), anti-CXCR4 (anti-CXCR4-PE, Beckman Coulter Company), anti-CD14, anti-CD86, and anti-HLA-DRII monoclonal antibodies (anti-CD14-alexa Fluor 750, anti-CD86-PC7, and anti-HLA-DRII-PC5.5, Beckman Coulter Company) [30 (link)].
Relevant isotype controls for each monoclonal antibody were used in the initial setup and frequently between tests.

Cutolo M., Soldano S., Montagna P., Trombetta A.C., Contini P., Ruaro B., Sulli A., Scabini S., Stratta E., Paolino S., Pizzorni C., Smith V, & Brizzolara R. (2018). Effects of CTLA4-Ig treatment on circulating fibrocytes and skin fibroblasts from the same systemic sclerosis patients: an in vitro assay. Arthritis Research & Therapy, 20, 157.

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Comprehensive Immune Cell Profiling by Flow Cytometry

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NK, T and Monocyte cell surface antigens were analysed by flow cytometry. Briefly, two million PBMCs were stained with a surface antibody’s cocktail: anti-CD3 PerCP-Cy 5.5, anti-CD56 APC and anti-CD16 PE-CyTM7 from BD Biosciences, anti-CD45 Krome Orange from Beckman Coulter and anti-Vδ2 Brilliant Violet 605 from Biolegend (Mix NK and gammadelta T cells); anti-CD4 FITC, anti-CD8 PE and anti-CD3 Pacific Blue from BD Biosciences and anti-CD45 Krome Orange (Mix T cells); anti-CD14 APC-H7 and anti-CD16 V450 from BD Biosciences and anti-CD45 Krome Orange (Mix Monocytes), in 1× PBS, 1% Bovine Serum Albumin (BSA) (from Sigma Aldrich) and 0.1% Sodium azide (NaN3) (from Serva) solution for 15 min at 4°C. Afterwards, PBMCs were washed with 0.3 mL of 1× PBS, 1% BSA and 0.1% NaN3 solution and centrifuged at 1600 rpm for 5 min. Next, PBMCs were fixed with 1% Paraformaldehyde (PFA) (Bio-Rad, Hercules, California, USA) in 1× PBS, 1% BSA and 0.1% NaN3 solution for 15 min at room temperature in the dark, washed as before and centrifuged at 1600 rpm for 5 min. Cells were acquired using a FACSLyric (BD Biosciences) and analysed by BD FACSuite software (BD Biosciences).

Bordoni V., Matusali G., Mariotti D., Antonioli M., Cimini E., Sacchi A., Tartaglia E., Casetti R., Grassi G., Notari S., Castilletti C., Fimia G.M., Capobianchi M.R., Ippolito G, & Agrati C. (2022). The interplay between SARS-CoV-2 infected airway epithelium and immune cells modulates regulatory/inflammatory signals. iScience, 25(2), 103854.

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Monocyte and Dendritic Cell Characterization

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After the 6 h incubation period, samples were aliquoted in two different tubes (0.250 mL/tube) in order to analyse the intracellular production of TNF-α in classical and non-classical monocytes as well as in mDCs. For the identification of these populations, cells were stained with the following monoclonal antibody combination: anti-CD45 krome orange (clone: J.33; Beckman Coulter – Immunotech, Marseille, France), anti-CD33 phycoerythrin cyanine 7 tandem (clone: D3HL60.251; Beckman Coulter – Immunotech) anti-CD14 allophycocyanin (clone: RM052; Beckman Coulter – Immunotech) and anti-HLA-DR peridinin chlorophyll protein cyanine 5 (clone: L243; Becton and Dickinson (BD) Biosciences, San Jose, CA, USA). After gentle mixing, cells were incubated for 15 min at room temperature in the dark followed by an intracytoplasmatic permeabilization protocol with IntraPrep Permeabilization Reagent (Beckman Coulter – Immunotech). Cells were fixed and permeabilized according to the manufacturer’s instructions. Thereafter, anti-TNF-α antibody (clone MAb11; BD Pharmingen, San Diego, CA, USA) was added and incubated for 15 min at room temperature in the dark. The cells were then washed twice with phosphate-buffered saline (Gibco BRL-life Technologies) and resuspended in 0.250 mL of this buffer.

Carvalheiro T., Gomes D., Pinto L.A., Inês L., Lopes A., Henriques A., Pedreiro S., Martinho A., Trindade H., Young H.A., da Silva J.A, & Paiva A. (2015). Sera from patients with active systemic lupus erythematosus patients enhance the toll-like receptor 4 response in monocyte subsets. Journal of Inflammation (London, England), 12, 38.

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Intracellular Cytokine Profiling of PBMCs

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Intracellular cytokine measurement was performed according to Körholz et al., 2021 (7 (link)). Briefly PBMCs (1x10⁶/ml) were left unstimulated or stimulated with 10 ng/ml PMA (Phorbol-12-Myristat13-Acetat) and 1µg/ml Calcium-Ionophore (both Merck KGaA, Darmstadt, Germany) under addition Brefeldin A (1µl/ml, BD-Biosciences San José, CA) for 12h overnight. Cells then were harvested and washed twice with PBS/1%FCS. For surface staining, cells were incubated with anti-CD45RO-PE (5µl/test) (Biolegend, San Diego, CA), anti-CD3-APC (2µl/test), anti-CD4-APC-AlexaFluor750 (5µl/test) and anti-CD45-Krome Orange (5µl/test) (Beckman Coulter, Krefeld, Germany) for 30 min at 4°C. After beeing washed twice with PBS/1% FCS, cells were fixed and permeabilized with 100µl Cytofix/Cytoperm™ (BD Biosciences, San José, CA) for 20 min at 4°C and then washed twice according to the manufacturers instructions. For intracellular staining, cells were incubated with anti-IFNγ-FITC (5µl/test), anti-IL4-PE Cy7 (5µl/test) (both Biolegend) and anti-IL-17A eFlour 450 (5µl/test) (eBioscience/Thermo Scientific) and anti-CD4 APC-AlexaFluor750 (2µl/test) (Beckman-Coulter, Krefeld,Germany) for 45min at 4°C. Cells were washed twice with Perm/Wash Buffer (BD Biosciences) and diluted in 500µl PBS/1% FCS. Analysis was performed on a BD LSR II.

Pietzsch L., Körholz J., Boschann F., Sergon M., Dorjbal B., Yee D., Gilly V., Kämmerer E., Paul D., Kastl C., Laass M.W., Berner R., Jacobsen E.M., Roesler J., Aust D., Lee-Kirsch M.A., Snow A.L, & Schuetz C. (2022). Hyper-IgE and Carcinoma in CADINS Disease. Frontiers in Immunology, 13, 878989.

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PBMC Stimulation and Flow Cytometric Analysis

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Peripheral blood mononuclear cells (PBMCs) from the patients and healthy controls were either left untreated or stimulated with interferon (IFN)-α (40 000 unit/ml; PBL Assay Science, Piscataway, New Jersey, USA) for 15 min at 37ºC. Cells were then fixed and permeabilized with PerFix-EXPOSE reagents (Beckman Coulter) according to the manufacturer's protocol and stained with anti-CD3-phycoerythrin-cyanin (PC7), anti-CD4-APC, anti-CD8-Pacific Blue, anti-CD45-Krome orange (all from Beckman Coulter) and anti-STAT-1-Alexa Fluor 488 (BD Biosciences) antibodies.
For the expression of interleukin (IL)-17, PBMCs from the patients and healthy controls were stimulated for 12 h with 40 ng/ml phorbol myristate acetate (PMA; Sigma, St Louis, Missouri, USA) and 10 -5 M ionomycin (Sigma) in the presence of 1μg/mL GolgiPlug (BD Biosciences). Cells were then fixed and permeabilized with PerFix-nc reagents (Beckman Coulter) according to the manufacturer's protocol and stained with anti-CD3-PC7 and anti-IL-17-Pacific Blue antibodies (Beckman Coulter). The measurement and analysis were carried out using flow cytometry (NAVIOS; Beckman Coulter) and Kaluza software (Beckman Coulter).

Shamriz O., Lev A., Simon A.J., Barel O., Javasky E., Matza-Porges S., Shaulov A., Davidovics Z., Toker O., Somech R., Zlotogorski A., Molho-Pessach V, & Tal Y. (2021). Chronic demodicosis in patients with immune dysregulation: An unexpected infectious manifestation of Signal transducer and activator of transcription (STAT)1 gain-of-function. Clinical and experimental immunology, 206(1).

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Multi-marker Phenotypic Analysis of Peripheral Blood Leukocytes

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EDTA anticoagulated peripheral blood was used for phenotype analysis by flow cytometry. In detail, 200 μL of whole blood was lysed in 2 mL of VersaLyse at room temperature for 15 min. Then, cells were washed twice with FACS buffer, followed by resuspension in the appropriate antibody preparation and incubated for 20 min at 4°C. For intracellular staining of Ki-67 expression, we used permeabilization and fixation method using eBioscience perm/fix kit (ebioscience cat no. 88-8824-00). The following monoclonal antihuman antibodies were used in appropriate concentrations to stain cells: anti-CD45-Krome orange (Beckman Coulter, cat no. 96416), anti-CD14-PC5.5 (Beckman Coulter, cat no. A70204), anti-CD 19-APC-Alexa fluor 750 (Beckman Coulter, cat no.A94681), anti-CD27-PE (BD Pharmingen, cat no.555441), anti-CXCR3-APC (BD Pharmingen, cat no. 561732), anti-CXCR4-APC (BD Pharmingen, cat no. 560936), anti-CD95-APC (BD Pharmingen, cat no. 558814), anti-ki-67-PE (Ebioscience Cat no. 12-5699-42), and anti- IgD-FITC (BD Pharmingen, cat no. 555778). After staining, the cells were analyzed by 10-color flow cytometry (Navios, Beckman Coulter), with at least 20,000 CD19+ events collected for each analysis.

Mahmood Z., Schmalzing M., Dörner T., Tony H.P, & Muhammad K. (2020). Therapeutic Cytokine Inhibition Modulates Activation and Homing Receptors of Peripheral Memory B Cell Subsets in Rheumatoid Arthritis Patients. Frontiers in Immunology, 11, 572475.

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