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Pe conjugated cd31 antibody

Manufactured by BD

The PE-conjugated CD31 antibody is a laboratory reagent used to identify and quantify CD31-positive cells in various biological samples. CD31, also known as PECAM-1, is a cell surface glycoprotein expressed on endothelial cells, platelets, and certain immune cells. This antibody is conjugated with the fluorescent dye Phycoerythrin (PE), allowing for the detection and analysis of CD31-expressing cells using flow cytometry or other fluorescence-based techniques.

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3 protocols using pe conjugated cd31 antibody

1

Isolation and Characterization of Neonatal Rat Cardiac Cells

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Cardiac cells were isolated from the ventricles of 2-day old neonatal rat hearts according to previously described methods [25 (link), 28 (link)]. Animals were sacrificed using humane standards approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University. Ventricles were harvested, minced, and enzymatically dissociated by serial digestion with trypsin and collagenase. Isolated cells were resuspended in DMEM/F-12 with 10% Fetal Bovine Serum and 10% Horse Serum, and pre-plated for 45 min at 37 °C to increase the fraction of cardiomyocytes in the cell suspension [28 (link), 29 (link)]. To assess cardiomyocyte purity, pre-plated cells were fixed and permeabilized according to the manufacturer instructions (BD Biosciences # 554714), then stained with cardiac troponin T (cTnT) primary antibody (Abcam ab45932) and Alexa Fluor-conjugated secondary antibody. To assess fraction of endothelial cells, pre-plated cells were washed with PBS and stained with PE-conjugated CD31 antibody (BD Biosciences #555027) and APC-conjugated CD90 antibody (BD Biosciences #561409). Stained cells were analyzed in Beckman Astrios Sorter and BD FACSCanto A analyzer at the Duke Cancer Institute’s Flow Cytometry Shared Resource. Endothelial cells and fibroblasts were identified as CD31+ and CD90+ /CD31 [30 (link)] cells, respectively.
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2

In Vivo Transduction of Endothelial Cells

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The EC71 and EC73 capsid genes were used to package CMV-GFP reporter vectors for comparison with AAV1 and AAV2 packaged vectors. A total of 3 × 1011 vector genomes of reporter vectors were injected into 7-week-old male C57BL/6J mice via the tail vein. After 2 weeks, the heart and liver were collected and digested in collagenase type I (GIBCO) at 37°C for 40 min, pipetted several times, and filtered through a 70-micron filter. After treatment with red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), cells were protected from light and incubated for 30 min on ice with 1% donkey serum in PBS containing both PE-conjugated CD31 antibody (BD PharMingen, 553373) and PerCP-Cy conjugated CD45 antibody (BD PharMingen, 550994) at a 1:300 dilution. Cells were then washed three times with PBS and fixed in 2% paraformaldehyde at 4°C. Finally, the cells were washed once with PBS and analyzed for the colocalization of GFP+ cells and ECs (CD31+, CD45) by an LSRFortessa flow cytometer (BD Biosciences). Analysis was performed using FlowJo vX.
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3

Piezo2 Knockout in Vascular and Neuronal Cells

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For Tie2Cre;Piezo2cKO and control mice, lungs were removed from adult mice (n = 4) and were processed into a single-cell suspension. Cells were then stained with PE-conjugated CD31 antibody (BD Pharmingen) for 30 min to 1 h on ice and were subjected to fluorescence-activated cell sorting (FACS). DAPI+ cells were excluded, and DAPI CD31+ and DAPI CD31 cells were sorted into separate tubes containing TRIzol. Total RNA was isolated for qRT–PCR. For Wnt1Cre;Piezo2cKO and control pups, DRG were removed from newborn pups (n = 5) and placed into TRIzol. Total RNA was isolated and subjected to qRT–PCR as previously described4 (link).
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