Cd45 krome orange clone j 33

Manufactured by Beckman Coulter

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CD45-Krome Orange (clone J.33) is a flow cytometry reagent that labels the CD45 antigen on the surface of human cells. CD45 is a transmembrane glycoprotein expressed on the majority of leukocytes. This reagent is designed for the identification and enumeration of leukocyte populations.

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CD45-Krome Orange Krome Orange

5 protocols using cd45 krome orange clone j 33

Multiparameter Analysis of Immune Cell Subsets

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Different cell populations were labeled with antibodies to CD45 (total leukocytes), CD14 and CD16 (monocytes), CD4 (T helper lymphocytes), CD28 (negative selection for autoreactive T helper lymphocytes), and CD69 (activated lymphocytes).
The antibodies used were the following: CD45-Krome Orange (clone J.33) (CD45-KO), CD4-PhycoerythrinTexas Red-X (Clone SFCI12T4D11 (T4)) (CD4− ECD), CD16-Allophycocyanin-Alexa Fluor 750 (clone 3G8) (CD16-APC-AlexaFluor750) from Beckman Coulter (Miami, FL, USA) and CD14-Pacific Blue (clone M5E2) (CD14-PB), CD28-Pacific Blue (clone CD28.2) (CD28-PB), CD69− Phycoerythrin (clone FN50) (CD69-PE).

Mangas-Losada A., García-García R., Leone P., Ballester M.P., Cabrera-Pastor A., Urios A., Gallego J.J., Martínez-Pretel J.J., Giménez-Garzó C., Revert F., Escudero-García D., Tosca J., Ríos M.P., Montón C., Durbán L., Aparicio L., Montoliu C, & Felipo V. (2019). Selective improvement by rifaximin of changes in the immunophenotype in patients who improve minimal hepatic encephalopathy. Journal of Translational Medicine, 17, 293.

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Characterizing T Regulatory Cells by Flow Cytometry

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Flow cytometry was performed on a FACSCanto II flow cytometer (BD Biosciences, USA).
Briefly, 50 μl of fresh whole blood was incubated with the appropriate amounts of fluorochrome-labelled monoclonal antibodies CD45 Krome Orange (clone J33), CD4 FITC (clone 13B8.2), CD25 APC (clone IHT44H3) and CD39 PC5.5 (clone BA54) from Beckman Coulter, France. Incubation was done at room temperature in the dark for 15 min using appropriate mouse immunoglobulin isotypes as a control. Following incubation, 1 ml erythrocyte lysing solution (VersaLyse, Beckman Coulter) was added to the samples and incubated under the same conditions for 20 min. Then, cells were fixed and permeabilized (using intraprep permeabilization reagent, Beckman Coulter), followed by intracellular staining with anti-FoxP3-PE (clone 259D, Beckman Coulter, France) for 30 min. Cells were resuspended in PBS and analysed. Finally, the cells were characterized by flow cytometry analysis using BD FACEDiva Software. Analysis performed with CD4 + CD25 + CD39+ FOXP3+ T-reg cells expressed as a percentage of the whole CD4 subset (Fig. 1).Fig. 1

A representative flowcytometric plot demonstrating FoxP3 versus CD25 and CD39 expression on CD4+ T cells

Samaan E., Elmaria M.O., Khedr D., Gaber T., Elsayed A.G., Shenouda R.N., Gamal H., Shahin D., Abousamra N.K, & Shemies R. (2022). Characterization of regulatory T cells in SARS-CoV-2 infected hemodialysis patients: relation to clinical and radiological severity. BMC Nephrology, 23, 391.

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Multicolor Flow Cytometry Immunophenotyping

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Different cell populations were labelled with antibodies to CD45 (total leukocytes), CD3 (T lymphocytes), CD4 (T helper lymphocytes), CD28 (negative selection for autoreactive T helper lymphocytes). The antibodies used were the following: CD45-Krome Orange (clone J.33) (CD45-KO), and CD4-PhycoerythrinTexas Red-X (Clone SFCI12T4D11 (T4)) (CD4- ECD), from Beckman Coulter (Miami, FL, USA); CD3- Allophycocyanin (clone UCHT1) (CD3-APC), and CD28-Pacific Blue (clone CD28.2) (CD28-PB) from Biolegend (San Diego, CA, USA).

Urios A., Ordoño F., García-García R., Mangas-Losada A., Leone P., José Gallego J., Cabrera-Pastor A., Megías J., Fermin Ordoño J., Felipo V, & Montoliu C. (2019). Tadalafil Treatment Improves Inflammation, Cognitive Function, And Mismatch Negativity Of Patients With Low Urinary Tract Symptoms And Erectile Dysfunction. Scientific Reports, 9, 17119.

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Multiparametric Flow Cytometry of Immune Cells

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Different cell populations were labeled with antibodies to CD45 (total leukocytes), CD14 and CD16 (monocytes), CD3 (T lymphocytes), CD4 (T helper lymphocytes), CD19 (B lymphocytes), CD28 (negative selection for autoreactive T helper lymphocytes), CD69 (activated lymphocytes) and CD45RA and CD45RO (naive and memory T lymphocytes).
The antibodies used were the following: CD45-Krome Orange (clone J.33) (CD45-KO), CD4-PhycoerythrinTexas Red-X (Clone SFCI12T4D11 (T4)) (CD4- ECD), CD19- Allophycocyanin-Alexa Fluor 700 (clone J3.119) (CD19-APC-AlexaFluor700) and CD16-Allophycocyanin -Alexa Fluor 750 (clone 3G8) (CD16-APC-AlexaFluor750) from Beckman Coulter (Miami, FL, USA) and CD14-Pacific Blue (clone M5E2) (CD14-PB), CD3- Allophycocyanin (clone UCHT1) (CD3-APC), CD28-Pacific Blue (clone CD28.2) (CD28-PB), CD69- Phycoerythrin (clone FN50) (CD69-PE), CD45RA- Peridinin Chlorophyll Protein/Cyanin5.5 (clone HI100) (CD45RA-PERCP/Cy5.5) and CD45RO-Allophycocyanin/Cyanin7 (clone UCHL1) (CD45RO- APC/Cy7) from Biolegend (San Diego, CA, USA).

Mangas-Losada A., García-García R., Urios A., Escudero-García D., Tosca J., Giner-Durán R., Serra M.A., Montoliu C, & Felipo V. (2017). Minimal hepatic encephalopathy is associated with expansion and activation of CD4+CD28−, Th22 and Tfh and B lymphocytes. Scientific Reports, 7, 6683.

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Multiparametric flow cytometry analysis of HSPC

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For each sample, 100 µl of peripheral EDTA blood was stained with monoclonal antibody cocktail: CXCR7 (FITC; clone 358426; R&D Systems), CXCR4 (PE-Cyanine7; clone 12G5; eBioscience), CD271 (PE, clone ME20.4-1.H4, Miltenyi Biotec), CD133/2 (APC, clone 293C3, Miltenyi Biotec), CD34 (eFluor450; clone 4H11; eBioscience), and CD45 (Krome Orange; clone J33; Beckman Coulter). Then, samples were lysed using lyse no-wash method with Immunoprep Reagent System and TQ-Prep Workstation (Beckman Coulter). The readout was done with Beckman Coulter Navios Flow Cytometer and a total count of 100,000 cells was acquired.
Samples were analyzed according to the SSc signal and CD45 expression and then the gate was put on the CD45 low to negative events (CD45 negative gate). Subsequently, the CD45 negative cells were analyzed as SSc low and the CD34 positive cells (CD45 negative/CD34 positive gate). Finally, expression of CXCR7, CXCR4, CD271, and CD133/2 was measured, in regard to the CD45 negative/the CD34 positive gate. The values were reported as mean fluorescence intensity (MFI). The analysis was done with Kaluza Software (Beckman Coulter).

Gójska-Grymajło A., Zieliński M., Gąsecki D., Kowalczyk K., Kwarciany M., Seroczyńska B, & Nyka W.M. (2018). CD271+, CXCR7+, CXCR4+, and CD133+ Stem/Progenitor Cells and Clinical Characteristics of Acute Ischemic Stroke Patients. Neuromolecular Medicine, 20(3), 301-311.

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