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Fitc conjugated cd45

Manufactured by BioLegend
Sourced in United States

FITC-conjugated CD45 is a fluorescently-labeled antibody that targets the CD45 cell surface antigen. CD45 is a tyrosine phosphatase expressed on the surface of most hematopoietic cells. The FITC (Fluorescein Isothiocyanate) fluorophore is conjugated to the CD45 antibody, allowing for the detection and identification of CD45-positive cells using flow cytometry.

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3 protocols using fitc conjugated cd45

1

Flow Cytometry Analysis of Mesenchymal Stem Cell Markers and Extracellular Vesicles

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FACS was performed using BD FACSAria III (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). To analyze the mesenchymal stem cell markers, 105 cells were stained with antibodies, particularly PE-conjugated CD105 (Bio-Rad, Hercules, CA, USA, MCA1557,), PE-conjugated CD90 (BioLegend, San Diego, CA, USA, 555596), PE-conjugated CD73 (BioLegend, 344004), FITC-conjugated CD45 (BioLegend, 555482), FITC-conjugated CD34 (BioLegend, 343504), and PE-conjugated CD14 (Bio-Rad, MCA1568). To determine the EV-specific marker, isolated EVs were incubated with 4% w/v aldehyde/sulfate-latex beads (Thermo Fisher Scientific, Rockford, IL, U.S, A37304) at room temperature overnight on rotation. Bead-binding extracellular cells were centrifugated at 1800× g for 10 min and washed with 500 µL of PBS. The pellet was resuspended with 50 µL of PBS containing CD63 (BioLegend, 353003) and CD81 (BioLegend, 349505) at 4 °C for 1 h. All antibodies were conjugated with PE fluorescence dye. Samples were washed with 500 µL of PBS and centrifuged at 1800× g for 10 min. The pellet was resuspended with PBS. Gating of exosome-decorated beads approximately 4 µm in diameter was analyzed using BD FACSAria III (BD Biosciences, San Jose, CA, USA). At least 10,000 events were acquired on flow cytometry. Data analyses was performed using BD FACSDiva™ software version 6.1.3 (BD Biosciences, San Jose, CA, USA).
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2

Isolation and Osteogenic Differentiation of BMSCs

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After the second passage, the cells were collected and identified using fluorescently-labeled antibodies for BMSC markers: Allophycocyanin-conjugated CD29 (cat. no. 102225; BioLegend, Inc.); FITC-conjugated CD44 (cat. no. 203906; BioLegend, Inc.); FITC-conjugated CD45 (cat. no. 202205; BioLegend, Inc.); and phycoerythrin-conjugated CD34 (ab223930; Abcam). Briefly, the cells were incubated with corresponding antibodies for 30 min at 4°C in the dark and then washed with PBS. The expression levels of different cell surface markers were detected using FACSCalibur flow cytometer and analyzed using CellQuestPro version 5.1.1 software (BD Biosciences).
After detecting the purity, BMSCs from the second passage were used in subsequent experiments. To induce osteogenic differentiation, BMSCs were initially cultured in complete culture media as aforementioned. When the cells reached 80% confluence, osteogenic-inducing medium [α-MEM (Cyagen Biosciences) supplemented with 10% FBS, 50 mg/ml ascorbate, 10 mM β-glycerophosphate, 100 nM dexamethasone and 1% penicillin-streptomycin] was used. Lastly, the plates were stained with alizarin red and alkaline phosphatase at four time-points (0, 3, 7, and 14 days).
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3

Immunophenotyping of Mesenchymal Stem Cells

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One million iMSCs and UCB-MSCs were stained with PE-conjugated CD73, PE-conjugated CD105, PE-conjugated CD90 (Miltenyi Biotec, Somerville, MA, USA), PE-conjugated CD34, and FITC-conjugated CD45 (BioLegend, San Diego, CA, USA) for 30 min at 4 °C. Stained cells were washed twice in PBS, and FACS was performed with a BD Accuri C6 flow cytometer (BD Biosciences, San Diego, CA, USA). The antibodies used are listed in Supplementary Table S6.
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